Novel aminoglycoside antibiotics

ABSTRACT

This invention relates to novel aminoglycoside antibiotics, which have potent antimicrobial activity against bacteria, which induce infectious diseases, particularly MRSA, and has no significant nephrotoxicity, and process for producing them. More particularly, the present invention relates to compounds represented by formula (Ia) or their pharmacologically acceptable salts or solvates, or their diastereomer mixtures, antimicrobial agents comprising them, and a process for producing them.

RELATED APPLICATION

The present application claims priority to Japanese Patent ApplicationNo. 155062/2006 filed on Jun. 2, 2006, the entire disclosure which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel aminoglycoside antibioticscharacterized in that they are effective against bacteria which induceclinically severe infectious diseases, particularly againstmethicillin-resistant Staphylococcus aureus (MRSA), and have a low levelof nephrotoxicity. The present invention also relates to novelintermediates useful for the production of the aminoglycosideantibiotics.

2. Background Art

In recent years, drug resistant bacteria resistant to antimicrobialagents used for the treatment of infectious diseases have appeared, andthe treatment of infectious diseases induced by the resistant bacteriahas become a major problem in clinical practice. In particular, MRSA isknown as one of major drug resistant bacteria, which rapidly propagatethrough hospital infection and induce clinically severe infectiousdiseases, and the development of therapeutic agents for the infectiousdiseases has been energetically made.

Aminoglycoside antibiotics have a broad antimicrobial spectrum fromgram-positive bacteria to gram-negative bacteria and have potentsterilizing power. Accordingly, the aminoglycoside antibiotics areexpected to function as promising medicaments which can overcome variousresistant bacteria including MRSA, and studies on the derivatives havebeen continuously carried out.

For example, Journal of Antibiotics, Vol. 24, 1971, p. 485 disclosesthat various derivatives of kanamycin, which is an aminoglycosideantibiotic, could be synthesized and 3′,4′-deoxykanamycin B (dibekacin)could be discovered from the kanamycin derivatives. Dibekacin is widelyused as a chemotherapeutic agent effective for resistant bacteria since1975.

Journal of Antibiotics, Vol. 26, 1973, p. 412 discloses(S)-1-N-(4-amino-2-hydroxybutyryl)dibekacin (arbekacin) obtained byacylating the amino group at 1-position of dibekacin with anaminohydroxybutyric acid. Further, Japanese Patent Publication No.10719/1988 discloses a production process of arbekacin.

Arbekacin has been used as a therapeutic agent for MRSA infectiousdiseases from the end of 1990. Arbekacin is known to have a broadantimicrobial spectrum from gram-positive bacteria including MRSA togram-negative bacteria including Pseudomonas aeruginosa. Ten years ormore have passed since arbekacin has become used as a therapeutic agentfor MRSA infectious diseases. Despite this fact, there is no reportabout a severely enhanced resistance. On the other hand, JAPANESESOCIETY OF CHEMOTHERAPY, Vol. 50, 2002, p. 494 reports that someclinically isolated MRSAs have lowered sensitivity to arbekacin.

Studies on various arbekacin analogues have been continuously carriedout. For example, WO 2005/070945 discloses that a group of compounds,characterized in that the steric configuration of the site correspondingto the 5-position of arbekacin has been inverted and varioussubstituents have been introduced, have antimicrobial activity againstMRSA.

On the other hand, nephrotoxicity is known, from long ago, as a sideeffect of aminoglycoside antibiotics. There is also a report aboutclinical influence of arbekacin (Japanese Patent Laid-Open No.164696/1980) on the kidney (JAPANESE SOCIETY OF CHEMOTHERAPY, Vol. 51,2003, p. 717).

In subject No. F-716 in the 44th Interscience Conference onAntimicrobial Agents and Chemotherapy (2004), the present inventorsdescribe, regarding an arbekacin analogue (compound No. TS2037:5,4″-diepiarbekacin) described in WO 2005/070945, the results of theevaluation of nephrotoxicity using proximal uriniferous tubuleepithelial cells of the kidney of pigs and usingβ-N-acetyl-D-glucosaminidase (hereinafter referred to as “NAG”) as anindex. The results show that the nephrotoxicity of the arbekacinanalogue is higher than that of arbekacin.

Studies on various methods for reducing the nephrotoxicity ofaminoglycoside antibiotics have hitherto been made. The combined use ofan aminoglycoside antibiotic and a compound for reducing thenephrotoxicity is reported as one of such methods. For example,fosfomycin is known to reduce the nephrotoxicity of some aminoglycosideantibiotics. Further, in a test using rats, there is a report that thecombined use of fosfomycin and arbekacin reduces the nephrotoxicity (TheJapanese Journal of Antibiotics, Vol. 47, 1994, p. 664).

A method using the so-called TDM (therapeutic drug monitoring), in whicha high level of therapeutic effect is realized while suppressing theside effect by making a medicine administration plan based onPharmacokinetics and Pharmacodynamics, has recently been studied. Forexample, there is a report that TDM is also utilized for anti-MRSAtreatment with arbekacin (JAPANESE SOCIETY OF CHEMOTHERAPY, Vol. 51,2003, p. 717).

When conventional aminoglycoside antibiotics, which are used clinically,are used solely, however, it is still required to reduce thenephrotoxicity while maintaining a broad antimicrobial spectrum andpotent antimicrobial activity against bacteria, which induce severeinfectious diseases, including MRSA. Accordingly, in aminoglycosideantibiotics, the development of a novel compound having a broadantimicrobial spectrum and potent sterilizing power and, at the sametime, having a low level of nephrotoxicity has been desired. Further,for the conventional aminoglycoside antibiotics, the appearance of drugresistant bacteria has become a problem, and compounds, which haveexcellent antimicrobial activity also against the drug resistantbacteria, have been still required. Furthermore, when the production ofexcellent antibiotics is taken into consideration, studies on stableproduction of the antibiotics are also a critical issue.

SUMMARY OF THE INVENTION

The present inventors have now succeeded in producing novel compoundsrepresented by formula (Ia) which has a broad antimicrobial spectrum andexcellent antimicrobial activity as well as a low level ofnephrotoxicity. The present inventors have further found that the novelcompound has a high level of antimicrobial activity against strainsfound in clinically isolated MRSAs, which possess a low level ofsensitivity against arbekacin.

The present inventors have further found a production process useful forstable production of the novel compounds, and an intermediate importantfor the production of the novel compounds.

The present invention has been made based on such finding.

Accordingly, an object of the present invention is to provide novelcompounds having a broad antimicrobial spectrum and excellentantimicrobial activity as well as a low level of nephrotoxicity, andsynthetic intermediates for the compounds.

According to the present invention, there is provided a compoundrepresented by formula (Ia) or its diastereomer mixture with respect tothe carbon atom attached with *, or their pharmacologically acceptablesalts or solvates.

wherein

R^(5ax) and R^(5eq), which may be the same or different, represent ahydrogen atom or hydroxyl,

R^(4″ax) and R^(4″eq), which may be the same or different, represent ahydrogen atom or hydroxyl,

n is an integer of 1 to 4, and

the configuration of carbon atom attached with * represents R or S.

Further, according to the present invention, there is provided anintermediate useful for the synthesis of the compound represented byformula (Ia).

The compound represented by formula (Ia) according to the presentinvention exhibits a broad antimicrobial spectrum and excellentantimicrobial activity and can realize the avoidance of severenephrotoxicity. Further, the compound represented by formula (Ia) canalso exhibit excellent antimicrobial activity against MRSAs having a lowlevel of sensitivity against arbekacin. The fact that the nephrotoxicityof the compound represented by formula (Ia) is lower than that ofarbekacin, is advantageous in the application of the compound, forexample, to patients suffering from infectious diseases. Accordingly,the compound represented by formula (Ia) according to the presentinvention can be advantageously utilized in the treatment of infectiousdiseases including MRSAs. Further, the compound represented by formula(Ia) can be stably supplied through compounds represented by formula(Xa), (Xb), or (XXV) which will be described later, and can beadvantageously utilized as a therapeutic agent for infectious diseases.

DETAILED DESCRIPTION OF THE INVENTION

The term “alkyl” as used herein as a group or a part of a group meansstraight chain, branched chain or cyclic alkyl unless otherwisespecified. The term “aryl” means phenyl or naphthyl unless otherwisespecified. The term “arylalkyl” means alkyl in which one or morehydrogen atoms are substituted by aryl.

Compounds Represented by Formula (Ia)

It is a feature of the compounds represented by formula (Ia) to have ahydroxyl group at its 2-position.

The compound having the above structure possesses a low level ofnephrotoxicity and, at the same time, possesses a broad antimicrobialspectrum of gram-positive bacteria including MRSAs to gram-negativebacteria including Pseudomonas aeruginosa and excellent antimicrobialactivity.

In the compounds represented by formula (Ia) in a preferred embodimentof the present invention, R^(5ax) and R^(5eq) are different from eachother and represent a hydrogen atom or hydroxyl, and R^(4″ax) andR^(4″eq) are different from each other and represent a hydrogen atom orhydroxyl.

In the compounds represented by formula (Ia), n is preferably 1 to 3,more preferably 1 to 2.

Further, in the compounds represented by formula (Ia) in a preferredembodiment of the present invention, the steric configuration of thehydroxyl group at 5-position is equatorial. Accordingly, in thecompounds represented by formula (Ia) in the above embodiment, R^(5ax)represents a hydrogen atom, and R^(5eq) represents hydroxyl. In thecompounds represented by formula (Ia) in a more preferred embodiment ofthe present invention, R^(4″ax) and R^(4″eq) are different from eachother, and represent a hydrogen atom or hydroxyl.

In a more preferred embodiment of the present invention, there areprovided compounds represented by formula (I) or their pharmacologicallyacceptable salts or solvates.

Further, in the compounds represented by formula (Ia) in anotherembodiment of the present invention, the steric configuration of thehydroxyl group at the 5-position is axial. Accordingly, in the compoundsrepresented by formula (Ia) in the above embodiment, R^(5ax) representshydroxyl, and R^(5eq) represents a hydrogen atom. In the compoundsrepresented by formula (Ia) in a more preferred embodiment of thepresent invention, R^(4″ax) and R^(4″eq) are different from each other,and represent a hydrogen atom or hydroxyl.

The compounds represented by formula (Ia) may exist as a salt. Examplesof such salts include pharmaceutically acceptable nontoxic salts.Specific examples thereof include hydrohalides such as hydrofluorides,hydrochlorides, hydrobromides, and hydroiodides, inorganic acid saltssuch as sulfates, nitrates, phosphates, perchlorates, and carbonates,salts of carboxylic acids such as acetic acid, trichloroacetic acid,trifluoroacetic acid, hydroxyacetic acid, lactic acid, citric acid,tartaric acid, oxalic acid, benzoic acid, mandelic acid, butyric acid,maleic acid, propionic acid, formic acid, and malic acid, salts of aminoacids such as alginic acid, aspartic acid, and glutamic acid, and saltsof sulfonic acid such as methanesulfonic acid and p-toluenesulfonicacid. Preferred are inorganic acid salts such as sulfates.

The compounds represented by formula (Ia) or their pharmacologicallyacceptable salts may exist as their solvates. Preferred solvates includehydrates or ethanolates.

As described above, the compounds represented by formula (Ia) may be theform of a diastereomer mixture with respect to the carbon atom attachedwith *, and the present invention includes this embodiment as well.

Synthetic Intermediates

The compounds represented by formula (Ia) may be produced by thefollowing two processes. According to these processes, the compoundsrepresented by formula (Ia) can be advantageously produced throughsynthetic intermediates which will be described later.

Synthetic intermediates in first production process

In the first production process according to the present invention,compounds represented by formula (Xa) and formula (Xb) are used assynthetic intermediates.

Accordingly, in one embodiment of the present invention, compoundsrepresented by formula (Xa) are provided.

wherein R^(5ax) and R^(5eq) are different from each other and representa hydrogen atom or hydroxyl.

In another embodiment of the present invention, there are providedcompounds represented by formula (Xb).

wherein R^(5ax) and R^(5eq) are different from each other and representa hydrogen atom or hydroxyl, and

R^(4″ax) and R^(4″eq) are different from each other and represent ahydrogen atom or hydroxyl.

In a preferred embodiment of the present invention, the compoundsrepresented by formula (Xb) are compounds represented by formula (XIV).

Synthetic Intermediates in Second Production Process According toPresent Invention

In the second production process according to the present invention,compounds represented by formula (XXV) or their diastereomer mixtureswith respect to the carbon atom attached with * are used as syntheticintermediates. In the compounds represented by formula (XXV) in thisproduction process, the steric configuration of the hydroxyl group at5-position is equatorial. Accordingly, the second production process issuitable for the production of compounds which are represented byformula (Ia) and of which the steric configuration of the hydroxyl groupat 5-position is equatorial.

wherein R² and G represent a protective group for hydroxyl group; R³,R^(2′), R^(6′) and E represent a protective group for amino group; n isan integer of 1 to 4; and the steric configuration of carbon atomattached with * represents R or S.

In the compounds represented by formula (XXV) in a more preferredembodiment of the present invention,

R² represents optionally substituted aryl C1-3 alkyl,

R³, R^(2′) and R^(6′), which may be the same or different, represent

-   -   optionally substituted C1-6 alkylsulfonyl,    -   optionally substituted arylsulfonyl, or    -   optionally substituted C1-6 alkyloxycarbonyl,

E represents optionally substituted C1-6 alkyloxycarbonyl, and

G represents a hydrogen atom,

-   -   optionally substituted C1-6 alkylcarbonyl, or    -   optionally substituted arylcarbonyl.

In the compounds represented by formula (XXV), aryl C1-3 alkyl grouprepresented by R² is preferably aryl C1-2 alkyl, more preferably benzyl.

One or more hydrogen atoms in the aryl C1-3 alkyl group represented byR² are optionally substituted, for example, by methoxy or nitro.Specific examples of substituted aryl C1-3 alkyl include methoxybenzylor nitrobenzyl.

In the compounds represented by formula (XXV), C1-6 alkylsulfonyl grouprepresented by R³, R^(2′) or R^(6′) is preferably C1-3 alkylsulfonyl,more preferably methanesulfonyl.

One or more hydrogen atoms in the C1-6 alkylsulfonyl group representedby R³, R^(2′) or R^(6′) are optionally substituted, for example, byoptionally substituted phenyl (phenyl or tolyl). Examples of substitutedC1-6 alkylsulfonyl groups include benzylsulfonyl or toluenesulfonyl.

One or more hydrogen atoms in the arylsulfonyl group represented by R³,R^(2′) or R^(6′) are optionally substituted, for example, by methyl.Specific examples of optionally substituted arylsulfonyl groups includebenzylsulfonyl or toluenesulfonyl.

In the compounds represented by formula (XXV), preferably, the C1-6alkyloxycarbonyl group represented by R³, R^(2′) or R^(6′) is C1-4alkyloxycarbonyl, more preferably methoxycarbonyl ortert-butoxycarbonyl.

One or more hydrogen atoms in the C1-6 alkyloxycarbonyl grouprepresented by R³, R^(2′) or R^(6′) are optionally substituted, forexample, by optionally substituted phenyl (for example, phenyl,methoxyphenyl, or nitrophenyl). Specific examples of the substitutedC1-6 alkyloxycarbonyl groups include benzyloxycarbonyl,tert-butoxycarbonyl, p-methoxybenzyloxycarbonyl orp-nitrobenzyloxycarbonyl.

In the compounds represented by formula (XXV), the C1-6 alkyloxycarbonylgroups represented by E is preferably C1-3 alkyloxycarbonyl, morepreferably methoxycarbonyl or ethoxycarbonyl, still more preferablymethoxycarbonyl.

One or more hydrogen atoms in the C1-6 alkyloxycarbonyl grouprepresented by E are optionally substituted, for example, by optionallysubstituted phenyl (for example, phenyl, methoxyphenyl, or nitrophenyl).Accordingly, specific examples of the C1-6 alkyloxycarbonyl groupsubstituted by optionally substituted phenyl include benzyloxycarbonyl,p-methoxybenzyloxycarbonyl, and p-nitrobenzyloxycarbonyl.

In the compounds represented by formula (XXV), the C1-6 alkylcarbonylgroup represented by G is preferably C1-3 alkylcarbonyl, more preferablyacetyl.

One or more hydrogen atoms in the C1-3 alkylcarbonyl group representedby G are optionally substituted, for example, by a halogen atom such aschlorine, bromine or fluorine, and specific examples of the substitutedC1-3 alkylcarbonyl group include trichloroacetyl and trifluoroacetyl.

In the compounds represented by formula (XXV), the arylcarbonyl grouprepresented by G is preferably benzoyl.

One or more hydrogen atoms in the arylcarbonyl group represented by Gare optionally substituted, for example, by phenyl, a halogen atom suchas chlorine, bromine or fluorine, nitro or methoxy. Specific examples ofthe substituted arylcarbonyl group include p-phenylbenzoyl,p-bromobenzoyl, p-nitrobenzoyl, and p-methoxybenzoyl.

In the compounds represented by formula (XXV), the aryl C1-3 alkyl grouprepresented by G is preferably aryl C1-2 alkyl, more preferably benzylor triphenylmethyl.

Further, one or more hydrogen atoms in the aryl C1-3 alkyl grouprepresented by G are optionally substituted, for example, by methoxy.Specific examples of the substituted C1-3 alkylsulfonyl group includep-methoxybenzyl.

In the compounds represented by formula (XXV), n is 1 to 4, preferably 1to 3, more preferably 1 or 2.

In the compounds represented by formula (XXV) in a more preferredembodiment of the present invention,

R² represents aryl C1-3 alkyl optionally substituted by methoxy ornitro,

R³, R^(2′) and R^(6′), which may be the same or different, represent

-   -   C1-6 alkylsulfonyl optionally substituted by optionally        substituted phenyl,    -   arylsulfonyl optionally substituted by methyl, or    -   C1-6 alkyloxycarbonyl optionally substituted by optionally        substituted phenyl,

E represents C1-6 alkyloxycarbonyl optionally substituted by optionallysubstituted phenyl, and

G represents a hydrogen atom,

-   -   C1-6 alkylcarbonyl,    -   arylcarbonyl, or    -   aryl C1-3 alkyl optionally substituted by methoxy.

In the compounds represented by formula (XXV) in a further preferredembodiment of the present invention,

R² represents aryl C1-2 alkyl optionally substituted by methoxy ornitro,

R³, R^(2′) and R^(6′), which may be the same or different, represent

-   -   C1-3 alkylsulfonyl optionally substituted by optionally        substituted phenyl,    -   arylsulfonyl optionally substituted by methyl, or    -   C1-4 alkyloxycarbonyl optionally substituted by optionally        substituted phenyl,

E represents C1-4 alkyloxycarbonyl optionally substituted by optionallysubstituted phenyl, and

G represents a hydrogen atom,

-   -   C1-3 alkylcarbonyl,    -   arylcarbonyl, or    -   aryl C1-2 alkyl optionally substituted by methoxy.

In the compounds represented by formula (XXV) in a further preferredembodiment of the present invention,

R² represents optionally substituted aryl C1-3 alkyl,

all of R³, R^(2′), R^(6′) and E represent optionally substituted C1-6alkyloxycarbonyl, and

G represents optionally substituted aryl C1-3 alkyl.

In the compounds represented by formula (XXV) in a further preferredembodiment of the present invention,

R² represents optionally substituted aryl C1-2 alkyl,

all of R³, R^(2′), R^(6′) and E represent optionally substituted C1-4alkyloxycarbonyl, and

G represents optionally substituted aryl C1-2 alkyl.

In the compounds represented by formula (XXV) in a further preferredembodiment of the present invention,

R² represents aryl C1-2 alkyl optionally substituted by methoxy ornitro,

all of R³, R^(2′), R^(6′) and E represent C1-4 alkyloxycarbonyloptionally substituted by phenyl optionally substituted by methoxy ornitro, and

G represents aryl C1-2 alkyl optionally substituted by methoxy.

In the compounds represented by formula (XXV) in another embodiment ofthe present invention,

R² represents benzyl, methoxybenzyl or nitrobenzyl,

R³, R^(2′) and R^(6′), which may be the same or different, representmethanesulfonyl, benzylsulfonyl,

p-toluenesulfonyl,

benzyloxycarbonyl, tert-butoxycarbonyl, p-methoxybenzyloxycarbonyl, orp-nitrobenzyloxycarbonyl,

E represents benzyloxycarbonyl, and

G represents a hydrogen atom,

-   -   acetyl,    -   benzoyl,    -   benzyl, p-methoxybenzyl, or triphenylmethyl.

In the compounds represented by formula (XXV) in a more preferredembodiment of the present invention,

R² represents benzyl, methoxybenzyl or nitrobenzyl,

all of R³, R^(2′), R^(6′) and E represent benzyloxycarbonyl, and

G represents benzyl.

Production Process

In the present invention, for example, the following two productionprocesses may be mentioned as production processes for compoundsrepresented by formula (Ia).

First Production Process

In the first production process according to the present invention,compounds represented by formula (Xa) and formula (Xb) are used assynthetic intermediates.

According to one aspect of the present invention, there is provided aprocess for producing a compound represented by formula (Ia):

wherein R^(5ax) and R^(5eq), which may be the same or different,represent a hydrogen atom or hydroxyl,

R^(4″ax) and R^(4″eq), which may be the same or different, represent ahydrogen atom or hydroxyl, and

n is an integer of 1 to 4,

the configuration of carbon attached with * represents R or S,

the process comprising the steps of

introducing protective groups into amino groups in a compoundrepresented by formula (Xa):

wherein R^(5ax) and R^(5eq) are as defined in formula (Ia),

reacting the compound represented by formula (Xa) with a compoundrepresented by formula (Xc):

wherein W represents a leaving group; Y^(ax) and Y^(eq), which may bethe same or different, represent group —OR^(4″) or a hydrogen atom;R^(2″), R^(4″) and R^(6″) represent a protective group for hydroxylgroup, and the configuration of carbon atom attached with * represents Ror S,

removing the protective groups from the resultant compound andconverting an azide group in the compound to an amino group to give acompound represented by formula (Xb):

wherein R^(5ax), R^(5eq), R^(4″ax) and R^(4″eq) are as defined informula (Ia),

optionally introducing protective groups into functional groups otherthan the amino group at 1-position of the compound represented byformula (Xb),

reacting the resultant compound with a compound represented by formula(XVII):

wherein E represents a protective group for amino group; G represents aprotective group for hydroxyl group; F represents a hydrogen atom or acarboxylic acid activating group; n is an integer of 1 to 4; and thesteric configuration of carbon atom attached with * represents R or S,and

removing the protective groups of the resultant compound to give thecompound represented by formula (Ia).

In the first production process according to the present invention, thecompound represented by formula (XVII) may be an enantiomer mixture withrespect to the carbon atom attached with *. Accordingly, according tothe first production process of the present invention, a diastereomermixture with respect to the carbon atom attached with * in the compoundrepresented by formula (Ia) can be produced. The present inventionincludes this embodiment as well.

In the first production process of the present invention, preferably,hydroxyl exists at 5- and 4″-positions in the compound represented byformula (Ia). Accordingly, in the first production process of thepresent invention in a preferred embodiment of the present invention,R^(5ax) and R^(5eq), which are different from each other, represent ahydrogen atom or hydroxyl; R^(4″ax) and R^(4″eq), which are differentfrom each other, represent a hydrogen atom or hydroxyl.

According to the first production process of the present invention, inthe compound represented by formula (Ia), when hydroxyl exists at5-position, the steric configuration of the hydroxyl group may beequatorial. Accordingly, in the first production process of the presentinvention in another preferred embodiment of the present invention,R^(5ax) represents a hydrogen atom, and R^(5eq) represents hydroxyl.

According to the first production process of the present invention, inthe compound represented by formula (Ia), when hydroxyl exists at5-position, the steric configuration of the hydroxyl group may be axial.Accordingly, in the first production process of the present invention inanother preferred embodiment of the present invention, R^(5ax)represents hydroxyl, and R^(5eq) represents a hydrogen atom.

In the first production process according to the present invention,preferably, the protective group introduced into the amino group of thecompound represented by formula (Xa) is introduced into 1-, 3-, 2′-, and6′-position of the compound represented by formula (Xa). Such protectivegroups include, for example, protective groups represented by R³,R^(2′), and R^(6′) in the compound represented by formula (XXV), orprotective groups represented by R¹, R³, R^(2′), and R^(6′) in scheme 2which will be described later. More specifically, protective groupsintroduced into the amino group at 1-, 3-, 2′-, and 6′-positions of thecompound represented by formula (Xa) are preferably protective groupscommonly used in synthetic organic chemistry, for example, optionallysubstituted alkylsulfonyl, optionally substituted arylsulfonyl, oroptionally substituted alkyloxycarbonyl, more preferably optionallysubstituted C1-6 alkylsulfonyl, optionally substituted arylsulfonyl, oroptionally substituted C1-6 alkyloxycarbonyl, still more preferably C1-6alkylsulfonyl optionally substituted by optionally substituted phenyl,arylsulfonyl optionally substituted by methyl, or C1-6 alkyloxycarbonyloptionally substituted by optionally substituted phenyl, furtherpreferably C1-3 alkylsulfonyl optionally substituted by optionallysubstituted phenyl, arylsulfonyl optionally substituted by methyl, orC1-4 alkyloxycarbonyl optionally substituted by optionally substitutedphenyl, still further preferably methanesulfonyl, benzylsulfonyl,p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl,p-methoxybenzyloxycarbonyl or p-nitrobenzyloxycarbonyl. More preferredis p-toluenesulfonyl or benzyloxycarbonyl.

The protective group introduced into a functional group other than theamino group at 1-position of the compound represented by formula (Xb) ispreferably introduced into the amino group at 2′-, 6′-, and 3″-positionsof the compound represented by formula (Xb). The protective groupintroduced into the 2′- and 6′-positions is the same as the protectivegroup introduced into the 2′- and 6′-positions of the compoundrepresented by formula (Xa). The protective group introduced into theamino group at 3″-position is the same as R^(3″) shown in scheme 3 whichwill be described later. Such protective groups may be the same as thosecommonly used in synthetic organic chemistry, preferably optionallysubstituted alkylcarbonyl, more preferably C1-3 alkylcarbonyl optionallysubstituted by a halogen atom, still more preferably trifluoroacetyl.

In the compounds represented by formula (Xc), the leaving grouprepresented by W is a halogen atom such as chlorine, bromine or iodine,alkylthio or arylthio, more preferably a halogen atom, C1-3 alkylthio orarylthio, still more preferably bromine or phenylthio.

The protective group for hydroxyl represented by R^(2″) is preferablyoptionally substituted arylalkyl, more preferably aryl C1-2 alkyloptionally substituted, for example, by nitro, still more preferablybenzyl, p-methoxybenzyl, or p-nitrobenzyl, further preferably benzyl.

The protective groups for hydroxyl represented by R^(4″) and R^(6″) maybe the same or different, and examples thereof include ester-typeprotective groups or ether-type protective groups, preferablyalkylcarbonyl, arylalkylcarbonyl, or optionally substituted arylalkyl,more preferably C1-6 alkylcarbonyl, aryl C1-3 alkylcarbonyl, or arylC1-3 alkyl optionally substituted by methoxy, more preferably acetyl,benzoyl, benzyl, p-methoxybenzyl, or triphenylmethyl.

The protective group for hydroxyl group represented by R^(4″) and R^(6″)together may form a cyclic protective group. The cyclic protective groupis preferably C3-8. Specific examples thereof include cyclic protectivegroups such as acetals or ketals, for example, cyclohexylidene acetal,isopropylidene acetal, or benzylidene acetal.

In the compounds represented by formula (XVII), for example, protectivegroups presented by formula (XXV) may be mentioned as the protectivegroup for amino group represented by E. More specifically, theprotective group for amino represented by E is preferably optionallysubstituted arylalkyloxycarbonyl, more preferably optionally substitutedaryl C1-6 alkyloxycarbonyl, still more preferably aryl C1-6alkyloxycarbonyl optionally substituted by optionally substitutedphenyl, further preferably benzyloxycarbonyl.

The carboxylic acid activation group represented by F is one used for areaction for forming a peptide bond by activating carboxyl (an activeesterification method), preferably a succinimide group, p-nitrophenyl,pentafluorophenyl or 1-hydroxybenzotriazole, more preferably asuccinimide group.

Further, the protective group for hydroxyl group represented by G maybe, for example, an ester-type protective group or an ether-typeprotective group, and examples thereof include protective groupsrepresented by formula (XXV). More specifically, the protective groupfor hydroxyl group represented by G is preferably optionally substitutedalkylcarbonyl, optionally substituted arylalkylcarbonyl, or optionallysubstituted arylalkyl, more preferably optionally substituted C1-6alkylcarbonyl, optionally substituted arylalkylcarbonyl, or optionallysubstituted aryl C1-3 alkyl, still more preferably C1-6 alkylcarbonyl;arylcarbonyl; or aryl C1-3 alkyl optionally substituted, for example, bymethoxy, further preferably, for example, acetyl, benzoyl, benzyl,p-methoxybenzyl, or triphenylmethyl.

Reaction conditions for each step in the first production processaccording to the present invention will be described in more detail in(1), (4), and (5) which will be described later.

Second Production Process

In the second production process according to the present invention, inaddition to the compounds represented by formula (Xa), the compoundsrepresented by formula (XXV) may be used as the synthetic intermediate.This process is suitable for the production of compounds, of which thesteric configuration of the hydroxyl group at 5-position is equatorial,among the compounds represented by formula (Ia).

According to another aspect of the present invention, there is provideda process for producing a compound represented by formula (Ia):

wherein

R^(5ax) represents a hydrogen atom,

-   -   R^(5eq) represents hydroxyl,    -   R^(4″ax) and R^(4″eq), which may be the same or different,        represent a hydrogen atom or hydroxyl,    -   n is an integer of 1 to 4, and    -   the steric configuration of carbon atom attached with *        represents R or S,    -   the process comprising the steps of

introducing protective groups into amino groups at the 3-, 2- and6′-positions and a hydroxyl group at 2-position of a compoundrepresented by formula (Xa):

wherein R^(5ax) and R^(5eq) are as defined in formula (Ia),

reacting the resultant compound with a compound represented by formula(XVII):

wherein E represents a protective group for amino group; G represents aprotective group for hydroxyl group; F represents a hydrogen atom or acarboxylic acid activating group; n is an integer of 1 to 4; and thesteric configuration of carbon atom attached with * represents R or S,

to give a compound represented by formula (XXV):

wherein R² represents a protective group for hydroxyl group; R³, R^(2′)and R^(6′) represent a protective group for amino group, and E, G, n andthe steric configuration of the carbon atom attached with * are asdefined in formula (XVII),

reacting the compound represented by formula (XXV) with a compoundrepresented by formula (Xc) or (Xd):

wherein W represents a leaving group; Y^(ax) and Y^(eq), which may bethe same or different, represent group —OR^(4″) or a hydrogen atom;R^(2″), R^(4″) and R^(6″) represent a protective group for hydroxylgroup; and the steric configuration of carbon atom attached with *represents R or S,

wherein W, Y^(ax), Y^(eq), R^(2″), R^(6″), and the steric configurationof carbon atom attached with * are as defined in (Xc), and R^(3″)represents a protective group for amino group, and

removing the protective groups from the resultant compound and, when thecompound represented by formula (Xc) is used, converting an azide groupin the compound to an amino group, to give a compound represented byformula (Ia).

In the second production process according to the present invention, thecompound represented by formula (XVII) may be an enantiomer mixture withrespect to the carbon atom attached with *. Accordingly, according tothe second production process of the present invention, a diastereomermixture with respect to the carbon atom attached with * in the compoundrepresented by formula (Ia) can be produced. The present inventionincludes this embodiment as well.

In the second production process of the present invention, preferably,hydroxyl exists at 4″-position in the compound represented by formula(Ia). Accordingly, in the second production process of the presentinvention in a preferred embodiment of the present invention, R^(4″ax)and R^(4″eq), which are different from each other, represent a hydrogenatom or hydroxyl.

In the second production process according to the present invention,specific embodiments of protective groups R², R³, R^(2′) and R^(6′)introduced into the amino groups at 3-, 2′- and 6-position and thehydroxyl group at 2-position are as described above. Further, W, Y^(ax),Y^(eq), R^(2″), and R^(6″) are the same as those in the first productionprocess.

Reaction conditions for each step in the second production processaccording to the present invention will be described in detail in (2)and (3) which will be described later.

Production Step of Synthetic Intermediate (Xa)

The compounds represented by formula (Ia) according to the presentinvention may be synthesized by using the compound represented byformula (II):O-3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1→6)-O-[2,6-diamino-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl-(1→4)]-D-streptamine (hereinafter referred to as“2-hydroxygentamicin C1a”) as one of starting materials for theproduction of the compounds. The compound represented by formula (II)can be advantageously used as starting materials for the production ofcompounds represented by formula (Xa) in the above two productionprocesses.

The compound represented by formula (II) (2-hydroxygentamicin C1a) is acompound obtained by substituting 2-deoxystreptamine, which is a partialconstituent element in gentamicin C1a as a gentamicin analogue, bystreptamine. This compound may be produced by a conventional method,that is, by adding an analogue of deoxystreptamine to a deoxystreptaminedependent producing strain of an aminoglycoside, culturing the producingstrain, and isolating a novel aminoglycoside antibiotic substance whichis a substance obtained by substituting a part of the constituentelement of the aminoglycoside by the added analogue from the resultantculture. More specifically, the compound represented by formula (II) maybe produced by adding streptamine to a deoxystreptamine dependentproducing strain of gentamicin C1a and culturing the mixture. Suchproducing strains include, for example, Micromonospora purpurea ATCC31119. The above production process is described in detail in JapanesePatent Laid-Open No. 108041/1976 the contents of which are incorporatedherein by reference. The process for producing the aminoglycosideantibiotic substance derivative and the method for obtaining a producingstrain for use in the production process are described, for example, inShier, W. T., K. L. Rinehart Jr. & D. Gottlieb et al., Proc. Nat. Acad.Sci. 63: pp. 198 to 204, (1969) in which neomycin is an object compound,and in Kojima M, Sato A et al., 3. Antibiot 26 (12): pp. 784-6 (1973) inwhich ribostamycin and kanamycin are object compounds.

In the production process according to the present invention, when acompound, which is represented by formula (Ia) and of which the hydroxylgroup at 5-position is equatorial, is produced, preferably, the compoundrepresented by formula (II) is hydrolyzed to produce a compoundrepresented by formula (Xa). Conditions for hydrolysis are described indetail in the first to sixth steps of scheme 2 which will be describedlater.

wherein R^(5ax) represents a hydrogen atom; and R^(5eq) representshydroxyl.

In the production process according to the present invention, when acompound, which is represented by formula (Ia) and of which the stericconfiguration of the hydroxyl group at 5-position is axial, is produced,the steric configuration of the hydroxyl group at 5-position of thecompound represented by formula (II) is inverted. Thus, in anotherembodiment of the present invention, the process for producing thecompound represented by formula (Ia) comprises introducing a protectivegroups into the hydroxyl groups other than the hydroxyl groups at 4″-and 5-positions, and the amino groups of the compound represented byformula (II),

inverting the steric configuration of the hydroxyl group at the5-position of the resultant compound,

removing protective groups of the resultant compound and hydrolyzing thecompound to give a compound represented by formula (Xa).

wherein R^(5ax) represents hydroxyl; and R^(5eq) represents a hydrogenatom.

In a preferred embodiment of the present invention, the productionprocess further comprises eliminating the hydroxyl group at the4″-position before or simultaneously with the inversion of the stericconfiguration of the hydroxyl group at 5-position. This step isdescribed in detail in step 5-3 to step 5-5 in scheme 10 which will bedescribed later.

The protective groups introduced into the hydroxyl groups other than thehydroxyl group at the 4″- and 5-positions, and the amino groups of thecompound represented by formula (II), that is, R¹, R², R³, R^(2′),R^(6′), R^(2′), and R^(3″) are as defined above.

The production processes according to the present invention will beclassified according to the steric configuration of the hydroxyl groupsat 5- and 4″-positions and the type of the production process and willbe described in more detail.

(1) Production of Compounds of which 5- and 4″-Positions are Equatorial:First Production Process

In the first production process according to the present invention,among the compounds represented by formula (Ia), compounds wherein bothR^(5ax) and R^(4″ax) represent a hydrogen atom, both R^(5eq) andR^(4′eq) represent hydroxyl, may be produced according to the followingthree schemes, that is, scheme 1 (step 1-1 to step 1-5a and step 1-5b),scheme 2 (step 1-6 to step 1-7) and scheme 3 (step 1-8 to step 1-14).

In scheme 1, the process for producing compounds represented by formula(Xc) used in the production process according to the present inventionwill be specifically described.

wherein W represents a leaving group; Y^(ax) represents a hydrogen atom;Y^(eq) represents group —OR^(4″); R^(2″), R^(4″) and R^(6″) represent aprotective group for hydroxyl group; and the steric configuration ofcarbon atom attached with * represents R or S.

Scheme 1

Scheme 1 describes step 1-1 to sep 1-5a and step 1-5b. In scheme 1,compounds represented by formula (Xc) are classified into compoundsrepresented by formula (VIII) and compounds represented by formula (IX)according to the type of the leaving group represented by W.

wherein B represents a leaving group containing a sulfur atom such asmethylthio, ethylthio, or phenylthio, preferably phenylthio; Xrepresents a halogen atom such as chlorine, bromine or iodine,preferably a bromine atom; R^(1″) represents a protective group forhydroxyl group, preferably, for example, an ester-type protective groupsuch as acetyl or benzoyl, more preferably acetyl; R^(2″) represents aprotective group for hydroxyl group, preferably a benzyl-type protectivegroup, which can be removed by a catalytic hydrogen reduction method,such as benzyl, p-methoxybenzyl, or p-nitrobenzyl, more preferablybenzyl; and

R^(4″) and R^(6″), which may be the same or different, eachindependently represent a protective group for hydroxyl, for example, anester-type protective group such as acetyl or benzoyl, or an ether-typeprotective group such as benzyl, p-methoxybenzyl, or triphenylmethyl,preferably an ester-type protective group such as acetyl or benzoyl, orR^(4″) and R^(6″) together represent a cyclic protective group such asacetal or ketal for simultaneously protecting two hydroxyl groups, forexample, cyclohexylidene acetal, isopropylideneacetal, or benzylideneacetal.

Step 1-1

Step 1-1 is a step in which a protective group is introduced into twohydroxyl groups at 4- and 6-positions of the compound represented byformula (III) to give the compound represented by formula (IV). Theprotective group is an acetal- or ketal-type protective group in whichR^(4″) and R^(6″) combine to form one protective group, preferably anisopropylidene group. This step is achieved by reacting the compoundrepresented by formula (III) with a ketone typified by acetone or anacetal typified by 2,2-dimethoxypropane in the presence of an acid.

Solvents usable in this step include, for example,N,N-dimethylformamide, methylene chloride, chloroform,1,2-dichloroethane, or ethyl acetate. Among them, N,N-dimethylformamideis preferred. Acids usable herein include p-toluenesulfonic acid,pyridinium p-toluene sulfonate, camphor sulfonic acid or hydrochloricacid. Among them, p-toluenesulfonic acid is preferred.

The reaction temperature is in the range of 20° C. to the refluxtemperature. The reaction time is, for example, 1 to 24 hr.

In this reaction, for example, when an acetal typified by2,2-dimethoxypropane is used, a method may also be adopted in which thereaction is carried out while removing the alcohol produced as aby-product by distillation under the reduced pressure from the reactionsystem to accelerate the reaction.

Step 1-2

Step 1-2 is a step of producing the compound represented by formula (V)by introducing a protective group (R^(2″)) into the hydroxyl group at2-position of the compound represented by formula (IV). This step isachieved by reacting the compound represented by formula (IV) withR^(2″)X, wherein R^(2″) represents, for example, benzyl, p-methoxybenzylor p-nitrobenzyl, and X represents, for example, chlorine, bromine oriodine, in the presence of a base.

Solvents usable in this step include pyridine, N,N-dimethylformamide,tetrahydrofuran, 1,4-dioxane, or methylene chloride. Among them,N,N-dimethylformamide is preferred. Bases usable herein includepyridine, lutidine, collidine, triethylamine, diisopropylethylamine,4-dimethylaminopyridine, sodium hydride, and potassium hydroxide. Amongthem, sodium hydride is preferred.

The reaction temperature is −20° C. to 50° C. The reaction time is 1 to24 hr.

Step 1-3

Step 1-3 is a step of removing the protective group at the 4- and6-positions of the compound represented by formula (V) to give acompound represented by formula (VI). This step is achieved by reactingthe compound represented by formula (V) with an acid.

Solvents usable in this step include tetrahydrofuran, diethyl ether,1,4-dioxane, methanol, methylene chloride, chloroform, acetic acid,water, or a mixed solvents composed of them. Among them, a mixed solventcomposed of acetic acid and water is preferred. Acids usable hereininclude acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuricacid, p-toluenesulfonic acid, or boron trichloride. Among them, aceticacid is preferred.

The reaction temperature is in the range of 0° C. to the refluxtemperature. The reaction time is 0.1 to 12 hr.

Step 1-4

Step 1-4 is a step of introducing a protective group into each of thehydroxyl group at the 4- and 6-positions of the compound represented byformula (VI) and converting the methoxy group at the 1-position of thecompound represented by formula (VI) to an acyloxy group to give acompound represented by formula (VII).

This step can be achieved, for example, by simultaneously reacting acarboxylic acid represented by R^(1″)OH such as acetic acid and an acidanhydride represented by R^(4″) ₂O (or R^(6″) ₂O) such as aceticanhydride with the compound represented by formula (VI) in the presenceof an acid catalyst.

Solvents usable in the above step include, for example, methylenechloride, chloroform, 1,2-dichloroethane, acetic acid, acetic anhydride,or a mixed solvent composed of them. Among them, a mixed solventcomposed of acetic acid and acetic anhydride is preferred. Acids usableherein include hydrogen chloride or sulfuric acid. Preferred is sulfuricacid.

The reaction temperature is −20° C. to 50° C. The reaction time is 1 to24 hr.

Step 1-4 can be carried out in two divided steps. In this case, at theoutset, a first step of introducing protective groups into the hydroxylgroups at the 4- and 6-positions is carried out, and a second step ofconverting the methoxy group at the 1-position to the acyloxy group iscarried out.

The first step is carried out by reacting a compound represented byformula (VI) with an acid anhydride such as acetic anhydride or an acidhalide such as acetyl chloride in the presence of a base. Solventsusable herein include, for example, pyridine, N,N-dimethylformamide,methylene chloride, chloroform, or 1,2-dichloroethane. Among them,pyridine is preferred. Bases usable herein include triethylamine,pyridine, or 4-dimethylaminopyridine. Among them, pyridine is preferred.The reaction temperature is −20° C. to 50° C. The reaction time is 1 to24 hr.

In the second step, the compound produced in the first step is reactedwith a carboxylic acid represented by R^(1″)OH such as acetic acid andan acid anhydride represented by R^(1″) ₂O such as acetic anhydride inthe presence of an acid catalyst. Solvents usable in the second stepinclude methylene chloride, chloroform, 1,2-dichloroethane, acetic acid,acetic anhydride, or a mixed solvent composed of them. Among them, amixed solvent composed of acetic acid and acetic anhydride is preferred.Acids usable herein include hydrogen chloride or sulfuric acid.Preferred is sulfuric acid.

The reaction temperature is −20° C. to 50° C. The reaction time is 1 to24 hr.

Step 1-5a

Step 1-5a is a step of converting the acyloxy group (OR^(1″)) at1-position of the compound represented by formula (VII) to a halogenatom to give a compound represented by formula (VIII). This step isachieved by reacting the compound represented by formula (VII) with ahydrogen halide represented by HX or a titanium halide represented byTiX₄ wherein X represents a chlorine atom or a bromine atom.

Solvents usable in this step include methylene chloride, chloroform,1,2-dichloroethane, ethyl acetate, or a mixed solvent composed of them.Among them, a mixed solvent composed of methylene chloride and ethylacetate is preferred.

The reaction temperature is −20° C. to 50° C. Further, the reaction timeis 1 to 24 hr.

Step 1-5b

Step 1-5b is a step of converting the acyloxy group (OR^(1″)) at1-position of the compound represented by formula (VII) to thioalkyl orthioaryl in the presence of a Lewis acid to give a compound representedby formula (IX). Specifically, this step is achieved by reacting thecompound represented by formula (VII) with a thiol represented by BH,wherein B represents, for example, methylthio, ethylthio, or phenylthio,or a trimethylsilylated thiol represented by TMS-B, wherein TMSrepresents trimethylsilyl, and B is as defined above, in the presence ofa Lewis acid.

Solvents usable in this step include, for example, methylene chloride,chloroform, 1,2-dichloroethane, ethyl acetate, or a mixed solventcomposed of them. Among them, methylene chloride is preferred. Lewisacids usable herein include trimethylsilyltriflate or tin chloride.Preferred is trimethylsilyltriflate. The reaction temperature is in therange of 20° C. to the reflux temperature. The reaction time is 1 to 48hr.

All the substituents at 1-position of the compound shown in scheme 1 cantake two steric configurations, that is, an axial form and an equatorialform. In scheme 1, these two steric configurations may be separated fromeach other before use in the reaction, or alternatively the two stericconfigurations in a mixed form as such may be used in the reaction.Further, the compound represented by formula (VIII) and the compoundrepresented by formula (IX) produced in scheme 1 may be separated intoan axial form and an equatorial form which are then used separately fromeach other in scheme 3. Alternatively, these compounds may be used as amixture of the axial form with the equatorial form.

Next, a process for producing a compound represented by formula (Xa)comprising step 1-6 and step 1-7 will be described in detail accordingto scheme 2. In scheme 2, the compound represented by formula (Xa) inwhich the steric configuration of the hydrogen atom at 5-position isequatorial corresponds to the compound represented by formula (X).

wherein R^(5ax) represents a hydrogen atom; and R^(5eq) representshydroxyl.

wherein

R¹, R³, R^(2′) and R^(6′) represents a protective group for amino group,preferably a protective group commonly used in organic syntheticchemistry such as methanesulfonyl, benzylsulfonyl, p-toluenesulfonyl,benzyloxycarbonyl, tert-butoxycarbonyl, p-methoxybenzyloxycarbonyl, orp-nitrobenzyloxycarbonyl, more preferably p-toluenesulfonyl orbenzyloxycarbonyl.

Step 1-6

Step 1-6 is a step for hydrolyzing the compound represented by formula(II) (2-hydroxygentamicin C1a) as a starting compound to give a compoundrepresented by formula (X). This step is achieved by heating thecompound represented by formula (II) in the presence of an acid.

In the above step, water is preferably used as a solvent. Acids usableherein include hydrochloric acid, sulfuric acid, nitric acid orhydrobromic acid. Among them, 3 to 5 M hydrochloric acid is preferred.

The reaction temperature is in the range of 20° C. to the refluxtemperature. The reaction time is 0.5 to 24 hr.

Step 1-7

Step 1-7 is a step of introducing protective groups into four aminogroups in the compound represented by formula (X) to give a compoundrepresented by formula (XI). This step can be achieved by reacting thecompound represented by formula (X) with a chloroformic ester such asbenzyl chloroformate, p-methoxybenzyl chloroformate, or p-nitrobenzylchloroformate, a carbonic diester such as di-tert-butyl dicarbonate, ora sulfonylating agent such as methanesulfonyl chloride, benzylsulfonylchloride, or p-toluenesulfonyl chloride in the presence of a base.

Solvents usable in this step include water, N,N-dimethylformamide,tetrahydrofuran, 1,4-dioxane, acetone, or a mixed solvent composed ofthem. Among them, a mixed solvent composed of water and 1,4-dioxane ispreferred. Bases usable herein include sodium hydroxide, potassiumcarbonate, sodium carbonate, triethylamine, pyridine, or4-dimethylaminopyridine. Preferred is sodium carbonate.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 24 hr.

The first production process according to the present invention will bedescribed in more detail according to scheme 3 which shows step 1-8 tostep 1-14. In scheme 3, the compound represented by formula (Ia) isfinally synthesized through a compound represented by formula (XIV)which is a key intermediate of the compound represented by formula (Ia).

wherein

B, X, R¹, R³, R^(2′), R^(6′), R^(2″), R^(4″), R^(6″), n, and the stericconfiguration of carbon atom attached with * are as described in schemes1 and 2,

E represents a protective group for amino, preferably a protective groupfor amino group commonly used in organic synthetic chemistry, morepreferably benzyloxycarbonyl,

F represents a hydrogen atom or a carboxylic acid activation group usedin a reaction which activates carboxyl to form a peptide bond (an activeesterification method), preferably a succinimide group, p-nitrophenyl,pentafluorophenyl, or 1-hydroxybenzotriazole, more preferablysuccinimide group,

G represents a hydrogen atom or a protective group for hydroxyl group,for example, an ester-type protective group such as acetyl or benzoyl,or an ether-type protective group such as benzyl, p-methoxybenzyl, ortriphenylmethyl, and

R^(3″) represents a protective group for amino group, preferably aprotective group for amino group commonly used in organic syntheticchemistry, more preferably trifluoroacetyl.

Step 1-8

Step 1-8 is a step of condensing the hydroxyl group at 6-position of thecompound represented by formula (XI) with the compound represented byformula (VIII) produced in step 1-5a or the compound represented byformula (IX) produced in step 1-5b to give a compound represented byformula (XII). This step can be achieved by reacting the compoundrepresented by formula (XI) with the compound represented by formula(VIII) or formula (IX) in the presence of a catalyst and a dehydratingagent.

Solvents usable in this step include, for example,N,N-dimethylformamide, methylene chloride, chloroform,1,2-dichloroethane, diethyl ether, or ethyl acetate, preferably1,2-dichloroethane. Catalysts usable herein includetrifluoromethanesulfonic acid, mercuric cyanide, N-iodosuccinimide,trifluoroacetic acid, mercury bromide, or yellow mercury oxide.Preferred is mercuric cyanide. Dehydrating agents usable herein include,for example, molecular sieves 4 A or Drierite, preferably Drierite.

The reaction temperature is −20° C. to 60° C. The reaction time is 1 to24 hr.

When X in formula (VIII) produced in step 1-5a is a bromine atom, thestep may be carried out by applying a conventional glycosylationreaction using a brominated succharide donor called a Koenigs-Knorr'sglycosylation reaction (Chem. Ber., Vol. 34, p. 957 (1901)). Conditionsfor this reaction may be properly determined by reference to a review ofH. Paulsen et al. (Angew. Chem. Int. Ed. Engl., Vol. 21, pp. 155-173(1982)), a review of R. R. Schmidt (Angew. Chem. Int. Ed. Engl., Vol.25, pp. 212-235 (1986)) and the like.

On the other hand, when B in formula (IX) produced in step 1-5b isthiophenyl, the step may be carried out by reference to a report of G.H. Veeneman et al. (Tetrahedron Letters, Vol. 31, pp. 1331-1334 (1990)),a report of P. Konradsson et al. (Tetrahedron Letters, Vol. 31, pp.4313-4316 (1990)) and the like.

Step 1-9

Step 1-9 is a step of removing the protective group (R^(4″)) at4″-position and the protective group (R^(6″)) at 6″-position of thecompound represented by formula (XII) to give a compound represented byformula (XIII). This step can be achieved by reacting the compoundrepresented by formula (XII) with a base.

Solvents usable in this step include methanol, ethanol, isopropylalcohol, tert-butyl alcohol, methylene chloride, chloroform, or a mixedsolvent composed of them. Among them, methanol is preferred. Basesusable herein include potassium carbonate, sodium carbonate, potassiumhydroxide, sodium hydroxide, sodium methoxide, sodium ethoxide, ortert-BuOK. Preferred is sodium methoxide.

The reaction temperature is −20° C. to 60° C., and the reaction time is1 to 24 hr.

Step 1-10

Step 1-10 is a step of producing the compound represented by formula(XIV) as the key intermediate. The compound represented by formula (XIV)constitutes a basic skeleton of the compound represented by formula(Ia). Accordingly, the compound represented by formula (XIV) may be usedas an intermediate for the production of the compound represented byformula (Ia) and its derivatives, and the present invention includesthis embodiment.

In step 1-10, all the protective groups in the compound represented byformula (XIII) are removed, and, further, the azido group at 3″-positionis converted to amino to give a compound represented by formula (XIV).This step can be achieved by radically reacting the compound representedby formula (XIII) with an alkali metal to remove the protective groupsfor all the amino groups and a hydroxyl group at 2″-position, andconverting the azido group at 3″-position to amino group, that is, byadopting the so-called “Birch reduction conditions.”

Solvents usable in the above step include liquid ammonia, methylamine,ethylamine, hexamethylphosphoamide, diethyl ether, tetrahydrofuran, or amixed solvent composed of them, preferably liquid ammonia. Alkali metalsusable herein include lithium, sodium, or potassium, preferably sodium.

The reaction temperature is −60° C. to 20° C., and the reaction time is0.5 to 24 hr.

When the protective group for amino group in the compound represented byformula (XIII) is a protective group which can be removed by a catalytichydrogen reduction reaction, for example, benzyloxycarbonyl,p-methoxybenzyloxycarbonyl, or p-nitrobenzyloxycarbonyl, the above stepcan also be carried out by reacting the compound represented by formula(XIII) with hydrogen in the presence of a catalytic hydrogen reductioncatalyst. Catalytic hydrogen reduction catalysts usable herein includepalladium-carbon, palladium black, palladium hydroxide, and platinumoxide. Among them, palladium-carbon is preferred. In the reaction, anysolvent may be used without particular limitation so far as the solventis not involved in the reaction. Preferred are methanol, ethanol,tetrahydrofuran, 1,4-dioxane, a mixed solvent composed of them, or amixed solvent composed of the above organic solvent and water.

The reaction temperature is 10° C. to 30° C. The reaction time isgenerally 1 to 8 hr.

When the protective group for amino in the compound represented byformula (XIII) is tert-butoxycarbonyl, a method may also be adopted inwhich the compound represented by formula (XIII) is reacted withhydrogen in the presence of a catalytic hydrogen reduction catalyst toremove the protective group for hydroxyl group at 2″-position and toconvert the azido group at 3″-position to amino group, followed by areaction of the resultant compound with an acid to removetert-butoxycarbonyl. In this case, solvents usable for removing theprotective group for amino group include ethyl acetate, methylenechloride, acetonitrile, acetone, anisole, water, or a mixed solventcomposed of them. Among them, water is preferred. Acids usable hereininclude p-toluenesulfonic acid, methanesulfonic acid, acetic acid, ortrifluoroacetic acid, preferably trifluoroacetic acid.

The reaction temperature is generally 0° C. to 30° C. The reaction timeis 1 to 12 hr.

Step 1-11

Step 1-11 is a step of selectively introducing protective group (R^(2′)and R^(6′)) into the amino groups at 2′ and 6′-positions of the compoundrepresented by formula (XIV) to give a compound represented by formula(XV). This step can be achieved by the compound represented by formula(XIV) with a chloroformic ester such as benzyl chloroformate,p-methoxybenzyl chloroformate, or p-nitrobenzyl chloroformate, acarbonic diester such as di-tert-butyl dicarbonate, or anN-(benzyloxycarbonyloxy)succinimide in the presence of a metal salt.

Solvents usable in this step include, for example,N,N-dimethylformamide, dimethylsulfoxide, methanol, ethanol, orisopropyl alcohol, preferably methanol. Transition metal salts usableherein include zinc acetate, nickel acetate, or cobalt acetate,preferably nickel acetate.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 24 hr.

Step 1-12

Step 1-12 is a step of selectively introducing a protective group(R^(3″)) into the amino group at 3″-position of the compound representedby formula (XV) to give a compound represented by formula (XVI). Thisstep can be achieved by reacting the compound represented by formula(XV), for example, with a halogenated carboxylic anhydride such astrifluoroacetic anhydride or trichloroacetic anhydride, a halogenatedcarboxylic ester such as methyl trifluoroacetate or ethyltrifluoroacetate, or an acid halide of a halogenated carboxylic acid.

In the above step, preferred reactants usable herein include ethyltrifluoroacetate.

Solvents usable in this step include, for example,N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran, or1,4-dioxane, preferably N,N-dimethylformamide.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 24 hr.

Step 1-13

Step 1-13 is a step of reacting the amino group at 1-position of thecompound represented by formula (XVI) with anω-amino-α-hydroxycarboxylic acid derivative represented by formula(XVII) to give a compound represented by formula (XVIII), that is, astep of conducting a peptide bond forming reaction. The compoundrepresented by formula (XVII) is, for example, a4-amino-2-hydroxybutyric acid derivative which can be prepared byconventional organic synthesis using a proper starting compound.Alternatively, the compound may be synthesized by reference to theprocess reported by H. Kawaguchi et al. (Journal of Antibiotics, Vol.25, pp. 695-708 (1972)). In this step, when a compound represented byformula (XVII) wherein F represents a hydrogen atom is used, a peptidecondensing agent commonly used in organic synthesis is used. Peptidecondensing agents include, for example, dicyclohexylcarbodiimide,diisopropylcarbodiimide, N-ethyl-N′-dimethyl aminopropylcarbodiimide andits hydrochloride, benzotriazol-1-yl-tris(dimethylamino)phosphoniumhexafluorophosphide, and diphenylphosphorylazide. They may be usedsolely, or alternatively may be used in combination withN-hydroxysuccinimide, 1-hydroxybenzotriazole or the like. When areaction which activates the carboxyl group to form a peptide bond (anactive esterification method) is used, in formula (XVII), F represents acarboxylic acid activation group selected from a succinimide group,p-nitrophenyl, pentafluorophenyl, 1-hydroxybenzotriazole or the like.That is, a compound called “active ester” is formed. In some cases, thisactive ester is isolated before use.

Solvents usable in the above step include N,N-dimethylformamide,dimethylsulfoxide, tetrahydrofuran, or 1,4-dioxane. Among them,tetrahydrofuran is preferred.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 48 hr.

Step 1-14

Step 1-14 is a step of removing the protective group in the compoundrepresented by formula (XVIII) to give a compound represented by formula(Ia) wherein both R^(5ax) and R^(4″ax) represent a hydrogen atom; andboth R^(5eq) and R^(4″eq) represent hydroxyl. This step can be achievedby reacting the compound represented by formula (XVIII) with a base toremove the protective group for amino group at 3″-position and thenreacting the resultant compound with hydrogen in the presence of acatalytic hydrogen reduction catalyst to remove the remaining protectivegroup for amino group.

Solvents usable in the above step of removing the protective group foramino at the 3″-position include methanol, ethanol, isopropyl alcohol,tert-butyl alcohol, tetrahydrofuran, 1,4-dioxane, water, or a mixedsolvent composed of them. Among them, a mixed solvent composed oftetrahydrofuran and water is preferred. Bases usable herein includeaqueous ammonia, potassium carbonate, sodium carbonate, potassiumhydroxide, or sodium hydroxide. Among them, aqueous ammonia ispreferred.

The reaction temperature is 0° C. to 50° C., and the reaction time is 1to 48 hr.

Catalytic hydrogen reduction catalysts usable in the step of removingthe remaining protective group for amino group other than the aminogroup at 3″-position include palladium-carbon, palladium black,palladium hydroxide, Raney nickel, or platinum oxide. Among them,palladium black is preferred. Any solvent may be used without particularlimitation so far as the solvent is inert to the reaction. Preferredsolvents include methanol, ethanol, tetrahydrofuran, 1,4-dioxane, aceticacid, a mixed solvent composed of them, or a mixed solvent composed ofthe organic solvent and water. A hydrogen gas may be used as hydrogenadded. The pressure of the hydrogen gas may be 1 atom which is theatmospheric pressure. If necessary, a pressurized hydrogen gas may alsobe used. Regarding hydrogen sources different from the hydrogen gas, ifnecessary, formic acid, a salt of formic acid, cyclohexene or the likemay also be used.

The reaction temperature is 10° C. to 30° C., and the reaction time isgenerally 1 to 8 hr.

When the protective group for amino group in the compound represented byformula (XVIII) is, for example, tert-butoxycarbonyl orp-methoxybenzyloxycarbonyl which can be removed under acidic conditions,the remaining protective group for amino group other than the aminogroup at 3″-position may also be removed by reacting the compound,produced by removing the protective group for amino at 3″-position, witha acid. In this case, solvents usable in the step of removing theprotective group for amino group include ethyl acetate, methylenechloride, acetonitrile, acetone, anisole, water, or a mixed solventcomposed of them. Among them, water is preferred. Acids usable hereininclude p-toluenesulfonic acid, methanesulfonic acid, acetic acid, ortrifluoroacetic acid. Among them, trifluoroacetic acid is preferred.

The reaction temperature is generally 0° C. to 30° C., and the reactiontime is 1 to 12 hr.

(2) Production of Compounds of which the 5- and 4″-Positions areEquatorial: Second Production Process

In the second production process according to the present invention,among the compounds represented by formula (Ia), compounds wherein bothR^(5ax) and R^(4″ax) represent a hydrogen atom and both R^(5eq) andR^(4″eq) represent hydroxyl, may be produced according to the followingscheme 4 (step 2-1 to step 2-7), scheme 5 (step 2-8 to step 2-10). Inthis case, the starting compound (X) is the same as the compoundsynthesized from the compound represented by (2-hydroxygentamicin C1a)represented by formula (II) in scheme 2.

wherein

R¹, R³, R^(2′), R^(6′), E, F, G, n, and the steric configuration of thecarbon atom attached with * are as defined in schemes 1 and 2; R²represents a protective group for hydroxyl group commonly used inorganic synthesis, preferably a benzyl-type protective group which canbe removed by a catalytic hydrogen reduction reaction such as benzyl,p-methoxybenzyl, or p-nitrobenzyl, more preferably benzyl; R⁵ and R⁶represent a protective group for hydroxyl group and each independentlyrepresent a protective group for hydroxyl group, or R⁵ and R⁶ togetherrepresent a cyclic protective group which simultaneously protects twohydroxyl groups, for example, an acetal or a ketal, preferablycyclohexylidene acetal.

In scheme 4, step 2-1 to step 2-6 are steps of introducing a protectivegroup into the amino groups at 3, 2′ and 6′-positions and the hydroxylgroup at 2-position of the compound represented by formula (X).

Step 2-1

Step 2-1 is a step of introducing an identical protective group intofour amino groups in the compound represented by formula (X) synthesizedin scheme 2 to give a compound represented by formula (XI). This stepcan be achieved by reacting the compound represented by formula (X) witha chloroformic ester such as benzyl chloroformate, p-methoxybenzylchloroformate, or p-nitrobenzyl chloroformate, a carbonic diester suchas di-tert-butyl dicarbonate, or N-(benzyloxycarbonyloxy)succinimide inthe presence of a base.

Solvents usable in this step include, for example, 1,2-dimethoxyethane,N,N-dimethylformamide, dimethylsulfoxide, methanol, ethanol, isopropylalcohol, tetrahydrofuran, 1,4-dioxane, diethyl ether, methylenechloride, chloroform, and water. They may be mixed together for use as amixed solvent. A mixed solvent composed of 1,2-dimethoxyethane or1,4-dioxane and water is preferred. Bases usable herein include organicbases such as triethylamine, diisopropylethylamine, N-methylmorpholine,and pyridine, and inorganic bases such as sodium carbonate, potassiumcarbonate, and sodium hydrogencarbonate. Among them, triethylamine ispreferred.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 24 hr.

Step 2-2

Step 2-2 is a step of introducing protective groups (R⁵ and R⁶) into thehydroxyl group adjacent to 5- and 6-positions of the compoundrepresented by formula (XI) to give a compound represented by formula(XX). The protective group for hydroxyl to be selected may be such thatR⁵ and R⁶ each independently serve as a protective group for hydroxylgroup. The protective group for hydroxyl group is preferably such thatR⁵ and R⁶ together form a cyclic protective group. Such protectivegroups include cyclohexylidene acetal, isopropylideneacetal, andbenzylidene acetal. In this scheme, cyclohexylidene acetal is preferred.

Solvents usable in this step include, for example,N,N-dimethylformamide, methylene chloride, chloroform,1,2-dimethoxyethane, 1,2-dichloroethane, ethyl acetate, or a mixedsolvent composed of them. Among them, 1,2-dimethoxyethane is preferred.Acids usable herein include p-toluenesulfonic acid, pyridinium p-toluenesulfonate, camphor sulfonic acid, or hydrochloric acid. Among them,pyridinium p-toluene sulfonate is preferred.

The reaction temperature is in the range of 20° C. to the refluxtemperature, and the reaction time is, for example, 1 to 24 hr.

In this reaction, when an acetal such as 2,2-dimethoxypropane orcyclohexanone dimethylacetal is used, the reaction may be carried outwhile removing the alcohol as a by-product from the reaction system bydistillation under the reduced pressure to accelerate the reaction.

Step 2-3

Step 2-3 is a step of introducing a protective group (R²) into thehydroxyl group at 2-position of the compound represented by formula (XX)to give a compound represented by formula (XXI). This step can beachieved by reacting the compound represented by formula (XX) with R²—X,wherein R^(2″) represents benzyl, p-methoxybenzyl, or p-nitrobenzyl, andX represents chlorine, bromine, iodine or the like, in the presence of abase.

Solvents usable in this step include pyridine, N,N-di methylformamide,tetrahydrofuran, 1,4-dioxane, or methylene chloride. Among them,N,N-dimethylformamide and tetrahydrofuran are preferred. Bases usableherein include pyridine, lutidine, collidine, triethylamine,diisopropylethylamine, 4-dimethylaminopyridine, sodium hydride, orpotassium hydroxide. Among them, sodium hydride is preferred.

In this reaction, for example, iodides such as sodium iodide andtetrabutylammonium iodide, silver salts such as silver oxide or silvernitrate, or crown ether may be added from the viewpoint of acceleratingthe reaction.

The reaction temperature is −20° C. to 50° C., and the reaction time is1 to 24 hr.

Step 2-4

Step 2-4 is a step of removing the protective groups for hydroxyl groupin R⁵ and R⁶ introduced in the step 2-3 to give a compound representedby formula (XXII). Reaction conditions used are those commonly used inorganic synthesis depending upon the protective groups introduced intoR⁵ and R⁶.

When the protective group for hydroxyl is such that R⁵ and R⁶ in formula(XXI) together form a cyclic protective group, this step can be achievedby reacting the compound represented by formula (XXI) with an acid.

Solvents usable in this step include tetrahydrofuran, diethyl ether,1,4-dioxane, methanol, methylene chloride, chloroform, acetic acid,water, or a mixed solvent composed of them. Among them, a mixed solventcomposed of chloroform and methanol or a mixed solvent composed ofacetic acid and water is preferred. Acids usable herein include aceticacid, trifluoroacetic acid, hydrochloric acid, sulfuric acid,p-toluenesulfonic acid, or boron trichloride. Among them,trifluoroacetic acid or acetic acid is preferred.

The reaction temperature is in the range of 0° C. to the refluxtemperature. The reaction time is 0.1 to 12 hr.

Step 2-5

Step 2-5 is a step of removing the protective group (R¹) for amino groupat 1-position of the compound represented by formula (XXI) and, at thesame time, forming a cyclic carbamate between the amino group and anadjacent hydroxyl group at 6-position to give a compound represented byformula (XXIII).

This step is achieved by treating the compound represented by formula(XXI) with a base. Bases usable herein include sodium hydride andpotassium hydroxide. Among them, sodium hydride is preferred. Solventsusable herein include N,N-dimethylformamide, tetrahydrofuran,1,4-dioxane, or methylene chloride. Among them, N,N-dimethylformamide ispreferred.

Step 2-6

Step 2-6 is a step of cleaving the cyclic carbamate formed between 1-and 6-position of the compound represented by formula (XXIII) to give acompound represented by formula (XXIV). This step can be achieved bytreating the compound represented by formula (XXIII) with a base.

Bases usable herein include inorganic bases such as sodium carbonate andpotassium carbonate, and metal alkoxides such as sodium methoxide andsodium ethoxide.

Solvents usable herein include N,N-dimethylformamide, tetrahydrofuran,1,4-dioxane, methylene chloride, water, and a mixed solvent composed ofthem. Among them, a mixed solvent composed of 1,4-dioxane and water ispreferred.

The reaction temperature is in the range of 0° C. to the refluxtemperature, and the reaction time is 0.1 to 12 hr.

Step 2-7

Step 2-7 is a step of condensing the amino group at 1-position of thecompound represented by formula (XXIV) produced in step 2-6 with thecompound represented by formula (XVII) to give a compound represented byformula (XXV).

This step can be achieved under the same reaction conditions as thereaction conditions described in step 1-13 or reaction conditionssimilar to the reaction conditions described in step 1-13 in scheme 3.

Scheme 5 shows the step of reacting the compound represented by formula(XXV) with the compound represented by formula (Xc) (step 2-8),

wherein W, Y^(ax), Y^(eq), R^(6′), R^(2″), and the steric configurationof carbon atom attached with * are as defined above, and

The step of removing the protective group from the resultant compound(step 2-9 and step 2-10) and the step of reducing the azido group of thecompound to give a compound represented by formula (Ia) (step 2-10) areexplained below.

wherein

R², R³, R^(2′), R^(6′), R^(2″), R^(4″), R^(6″), B, X, E, G, n, and thesteric configuration of carbon atom attached with * are as definedabove.

Step 2-8

Step 2-8 is a step of condensing the compound represented by formula(XXV) produced in scheme 4 with the compound represented by formula (Xc)(the compound represented by formula (VIII) or the compound representedby formula (IX)) to give a compound represented by formula (XXVI).

This step can be achieved under the same reaction conditions as thereaction conditions described in step 1-8 or reaction conditions similarto the reaction conditions described in step 1-8 in scheme 3.

Step 2-9

Step 2-9 is a step of removing two protective groups (R^(4″) and R^(6″))for hydroxyl group in the compound represented by formula (XXVI) to givea compound represented by formula (XXVII).

This step can be achieved by hydrolysis under basic conditions or underacidic conditions, or by treating the compound with a nucleophilic base,for example, sodium methoxide or sodium ethoxide under anhydrousconditions.

In the case of hydrolysis, in addition to water, alcohol solvents suchas methanol or ethanol, and solvents miscible with water, for example,with tetrahydrofuran or 1,4-dioxane may be used as the reaction solvent.Further, solvents immiscible with water, for example, ethyl acetate andmethylene chloride, may be used by adopting a two layer-type reactionwith water. Solvents commonly used in organic synthesis may be used asthe solvent under anhydrous conditions.

Step 2-10

Step 2-10 is a step of reducing the azido group in the compoundrepresented by formula (XXVII) produced in step 2-9 to amino group andfurther removing three protective groups (R³, R^(2′), and R^(6′)) foramino group and the protective group (R²) for hydroxyl group at2-position to give a compound represented by formula (XIX) which is acompound represented by formula (Ia) wherein both R^(5ax) and R^(4″ax)represent a hydrogen atom and both R^(5eq) and R^(4″eq) representhydroxyl.

In this reaction, the reduction reaction of the azido group and thedeprotection may be carried out separately from each other through aplurality of stages. Alternatively, when both the reactions can becarried out under the same reaction conditions, this step may be carriedout by a single reaction.

A reaction with hydrogen in the presence of a catalytic hydrogenreduction catalyst may be mentioned as the reaction through which theazido group is reduced and converted to amino group. Catalytic hydrogenreduction catalysts usable herein include palladium-carbon, palladiumblack, palladium hydroxide, Raney nickel, or platinum oxide. Among them,palladium black is preferred. Any solvent may be used without particularlimitation so far as the solvent is inert to this reaction. Preferredsolvents include methanol, ethanol, tetrahydrofuran, dioxane, aceticacid, a mixed solvent composed of them, or a mixed solvent composed ofthe organic solvent and water. A hydrogen gas may be used as hydrogen tobe added. The pressure of the hydrogen gas may be 1 atm which is theatmospheric pressure. If necessary, pressurized hydrogen gas may also beused. Hydrogen sources different from the hydrogen gas, for example,formic acid, salts of formic acid, and cyclohexene may also be usedaccording to need.

Methods for reducing the azido group for conversion to amino groupinclude the method of Staudinger et al. in which the azido compound isreacted with phosphine or phosphite to give iminophosphorane which isthen hydrolyzed for conversion to amino (Helvetica Chemica Acta, Vol. 2,p. 635 (1919)). Phosphine reagents usable in this method includetriphenylphosphine and trimethylphosphine. For example, trimethylphosphite may be mentioned as the phosphite reagent. Solvents usable inthe reaction include tetrahydrofuran, 1,4-dioxane, diethyl ether,acetonitrile, methylene chloride, water, and a mixed solvent composed ofthem. Iminophosphorane produced as an intermediate may be isolatedbefore hydrolysis. The production of iminophosphorane and hydrolysis canbe carried out in one step by adding water to a reaction solvent.

The method for removing the three protective groups (R³, R^(2′), andR^(6′)) for amino group and the protective group (R²) for hydroxyl groupat 2-position may be selected by taking into consideration deprotectionconditions commonly used in organic synthesis depending upon the type ofthe protective group. The method may be carried out stepwise or in onestep.

When a protective group, which can remove by a catalytic hydrogenationreaction, is selected for both the three protective groups (R³, R^(2′),and R^(6′)) for amino group and the protective group (R²) for hydroxylgroup at 2-position and, in this case, when all of R³, R^(2′), andR^(6′) represents benzyloxycarbonyl while R² represents benzyl, areduction reaction for converting the azido group to amino group canalso be advantageously simultaneously achieved by a one-step catalytichydrogenation reaction.

(3) Production of Compounds of which the 5- and 4″-Positions areEquatorial: Second Production Process

In the second production process according to the present invention, thecompound represented by formula (Ia) may also be produced by using thecompound represented by formula (Xd) instead of the compound representedby formula (Xc).

wherein W, Y^(ax), Y^(eq), R^(2″), R^(3″), R^(6″), and the stericconfiguration of carbon atom attached with * are as defined above.

The second production process will be described with reference to scheme6 (step 3-1 to step 3-4) and scheme 7 (step 3-5 to step 3-7).

In the following scheme 6, compounds represented by formula (Xd),wherein Y^(ax) represents a hydrogen atom, Y^(eq) represents hydroxyl,and W represents a leaving group (B), are described as compoundsrepresented by formula (XXXI), and the production process thereof willbe described.

wherein

R^(2″), R^(3″), R^(4″), R^(6″), and B are as defined above; and R^(4″)and R^(6″) preferably represent an ester-type protective group.

Step 3-1

Step 3-1 is a step of removing the two protective groups (R^(4″) andR^(6″)) for hydroxyl group in the compound represented by formula (IX)to give a compound represented by formula (XXVIII).

When R^(4″) and R^(6″) represent an ester-type protective group, ahydrolysis reaction or a solvolysis reaction can be applied. Morepreferred is a solvolysis reaction.

More specifically, the solvolysis reaction may be carried out usingsodium methoxide, sodium ethoxide or the like. Any solvent may be usedwithout limitation so far as the solvent is inert to the reaction.Preferred are alcohol solvents such as methanol and ethanol.

The reaction temperature is in the range of −20° C. to the refluxtemperature, and the reaction time is 0.1 to 12 hr.

Step 3-2

Step 3-2 is a step of reducing the azido group in the compoundrepresented by formula (XXVIII) to amino group to give a compoundrepresented by formula (XXIX).

In step 2-10 in scheme 5, the step of reducing the azido group to aminogroup is described in detail. The same conditions can be applied to step3-2. In scheme 6, preferably, the method of Staudinger et al. (HelveticaChemica Acta, Vol. 2, p. 635 (1919)) is used. The compound representedby formula (XXIX) produced in this step may be isolated for use in step3-3. Alternatively, the compound as such may be subsequently used as astarting compound for step 3-3 without isolation.

Step 3-3

Step 3-3 is a step of protecting an amino group of the compoundrepresented by formula (XXIX) to give the compound represented byformula (XXX).

Protective groups for amino group commonly used in organic synthesis maybe used as the protective group. Preferably, the protective group can beone which can be removed by a catalytic hydrogenation reaction.Specifically, benzyl-type protective groups such as benzyloxycarbonylmay be mentioned as the protective group. The protective group may beintroduced, for example, by a method described in connection with step1-7 in scheme 2, a method using a chloroformic ester described inconnection with step 2-1 in scheme 4, or a method using abenzyloxycarbonylation reagent such as anN-(benzyloxycarbonyloxy)succinimide group.

Step 3-4

Step 3-4 is a step of simultaneously protecting the hydroxyl group at4-position and the hydroxyl group at 6-position in the compoundrepresented by formula (XXX) to give a compound represented by formula(XXXI). R^(4″) and R^(6″) in scheme 6 preferably represent an ester-typeprotective group, particularly preferably acetyl.

The ester-type protective group can be introduced by a method commonlyused in organic synthesis using the acid anhydride or the acid halide inthe presence or absence of a base. Any solvent may be used withoutparticular limitation so far as the solvent is inert to the reaction.Preferred is a solvent which can serves both as a solvent and a basesuch as pyridine.

The reaction temperature is in the range of −20° C. to the refluxtemperature, and the reaction time is 0.1 to 72 hr.

Next, scheme 7 shows the step of reacting the compound represented byformula (XXV) with the compound represented by formula (XXXI) (step 3-5)and the step of removing the protective group of the resultant compoundto give a compound represented by formula (XIX) (step 3-6 and 3-7).

wherein R², R³, R^(2′), R^(6′), R^(2″), R^(3″), R^(4″), R^(6″), B, E, G,n, and the steric configuration of carbon atom attached with * are asdefined above.

Step 3-5

Step 3-5 is a step of condensing the compound represented by formula(XXV) produced in scheme 4 with the compound represented by formula(XXXI) in scheme 6 to give a compound represented by formula (XXXII).

Specifically the condensation can be carried out in the same manner asin step 2-8 in scheme 5.

Step 3-6

Step 3-6 is a step of removing the two protective groups (R^(4″) andR^(6″)) for hydroxyl group in the compound represented by formula(XXXII) produced in step 3-5 to convert the compound represented byformula (XXXIII).

Specifically, this step can be carried out in the same manner as in step2-9 in scheme 5.

Step 3-7

Step 3-7 is a step of removing all the protective groups (R², R³,R^(2′), R^(6′), R^(2″), R^(3″), E, and G) in the compound represented byformula (XXXIII) produced in step 3-6 to give a compound represented byformula (XIX) which is a compound represented by formula (Ia) in whichboth R^(5ax) and R^(4″ax) represent a hydrogen atom and both R^(5eq) andR^(4″eq) represent hydroxyl.

All the protective groups (R², R³, R^(2′), R^(6′), R^(2″), R^(3″), E,and G) in the compound represented by formula (XXXIII) can be removedstepwise or in one step (if possible) under deprotection conditions inconventional organic synthesis. For example, when all the protectivegroups in the compound represented by formula (XXXIII) can be removed bya catalytic hydrogenation reaction, the protective groups can be removedin one step by applying the same catalytic hydrogenation reactionconditions as in step 1-14 in scheme 3 or conditions similar to thecatalytic hydrogenation reaction conditions in step 1-14 in scheme 3.

(4) Production of Compounds of which the 5-Position is Equatorial andthe 4″-Position is Axial: Second Production Process

Further, according to the second production process of the presentinvention, compounds represented by formula (Ia), wherein both R^(5ax)and R^(4″eq) represent a hydrogen atom and both R^(5eq) and R^(4″ax)represent hydroxyl, can be produced according to scheme 8 (step 4-1 tostep 4-5a and step 4-5b) and scheme 9.

The process for producing compounds represented by formula (Xc) will bedescribed in detail with reference to scheme 8.

wherein W represents a leaving group; Y^(ax) represents group —OR^(4″);Y^(eq) represents a hydrogen atom; R^(2″), R^(4″) and R^(6″) represent aprotective group for hydroxyl group; and the steric configuration ofcarbon atom attached with * represents R or S.

In scheme 8, the compounds represented by formula (Xc) are classifiedaccording to the type of the leaving group represented by W intocompounds represented by formula (XXXVIII) and compounds represented byformula (XXXIX).

wherein

R^(2″) represents a protective group for hydroxyl group, preferably agroup selected from benzyl ether-type protective groups such as benzyland p-methoxybenzyl; R^(4″) and R^(6″), which are different from eachother, represent a protective group for hydroxyl group, for example, areselected from ester-type protective groups such as acetyl and benzoyl,and sulfonyl-type protective groups such as p-toluenesulfonyl,methanesulfonyl, and trifluoromethanesulfonyl; and B and X are asdefined above.

Step 4-1

Step 4-1 is a step of selectively introducing a protective group(R^(6″)) into only the hydroxyl group located at 6-position in the twohydroxyl groups in the compound represented by formula (VI) in scheme 1to give the compound represented by formula (XXXIV).

The protective group introduced is preferably a structurally bulkyprotective group among protective groups used as a protective group forhydroxyl group in organic synthesis, specifically preferably benzoyl orsubstituted benzoyl.

The reaction may be carried out in the same manner as in step 3-4 inscheme 6.

Step 4-2

Step 4-2 is a step of introducing a leaving group into the hydroxylgroup at 4-position of the compound represented by formula (XXXIV) togive a compound represented by formula (XXXV) which is a startingcompound in step 4-3 for synthesizing a compound represented by formula(XXXVI) in which the hydroxyl group at 4-position has been inverted.

A leaving group having a higher leaving ability than the protectivegroup introduced in R^(6″) in step 4-2 among leaving groups for hydroxylgroup in organic synthesis is selected as the leaving group introduced.Specifically, a sulfonyl-type leaving group is selected. Morespecifically, for example, R^(4″) in formula (XXXV) is preferablytrifluoromethanesulfonyl.

The reaction can be carried out in the same manner as in step 3-4 inscheme 6.

Step 4-3

Step 4-3 is a step of utilizing the leaving group at 4-position of thecompound represented by formula (XXXV) to give a compound represented byformula (XXXVI) of which the steric configuration at 4-position has beeninverted.

R^(4″) in the compound represented by formula (XXXVI) is preferably aprotective group which serves as a protective group for hydroxyl groupat 4-position and can be removed in the same manner as in R^(6″) as theprotective group for hydroxyl group at 6-position. R^(4″) is anester-type protective group, preferably acetyl.

The reaction which can provide the compound represented by formula(XXXVI) can be achieved by reacting the metal salt of a carboxylic acidwith the compound represented by formula (XXXV). The acetyl group whichis preferred as R^(4″) can be successfully introduced by using a salt ofacetic acid such as cesium acetate.

Any solvent may be used without particular limitation so far as thesolvent is inert to this reaction. The solvent is preferablyN,N-dimethylformamide.

The reaction temperature is in the range of −20° C. to refluxtemperature, and the reaction time is 0.1 to 72 hr.

Step 4-4

Step 4-4 is a step of converting the methoxy group at 1-position of thecompound represented by formula (XXXVI) to acyloxy (OR^(1″)) to give thecompound represented by formula (XXXVII).

This step can be achieved in the same manner as in step 1-4 in scheme 1.

Step 4-5a

Step 4-5a is a step of converting the acyloxy group (OR^(1″)) at1-position of the compound represented by formula (XXXVII) to a halogenatom to give a compound represented by formula (XXXVIII).

This step can be carried out in the same manner as in step 1-5a inscheme 1.

Step 4-5b

Step 4-5b is a step of converting the acyloxy group (OR^(1″)) at1-position of the compound represented by formula (XXXVII) to thioalkylor thioaryl in the presence of Lewis acid to give a compound representedby formula (XXXIX).

This step can be carried out in the same manner as in step 1-5b inscheme 1.

The substituent at 1-position of the compound shown in scheme 8 can taketwo steric configurations, that is, an equatorial form and an axialform. In scheme 8, they may be separated from each other before use inthe reaction, or alternatively the two steric configurations in a mixedform as such may be used in the reaction. Further, the compoundrepresented by formula (XXXVIII) and the compound represented by formula(XXXIX) produced in scheme 8 may be separated into an axial form and anequatorial form which are then used separately from each other in scheme9. Alternatively, these compounds may be used as a mixture of the axialform with the equatorial form.

wherein

R², R³, R^(2′), R^(6′), R^(2″), R^(4″), R^(6″), B, E, G, X, n, and thesteric configuration of the carbon atom attached with * are as definedabove.

Step 4-6

Step 4-6 is a step of condensing the compound represented by formula(XXV) produced in scheme 4 with the compound represented by formula(XXXVIII) or formula (XXXIX) in scheme 8 to introduce a compoundrepresented by formula (XXXX).

The condensation can be carried out in the same manner as in step 2-8 inscheme 5.

Step 4-7

Step 4-7 is a step of removing two protective groups (R^(4″) and R^(6″))for hydroxyl group in the compound represented by formula (XXXX)produced in the step 4-6 and converting to the compound represented byformula (XXXXI).

Specifically, this step can be carried out in the same manner as in step2-9 in scheme 5.

Step 4-8

Step 4-8 is a step of removing all the protective groups (R², R³,R^(2′), R^(6′), R^(2″), E, and G) in the compound represented by formula(XXXXI) produced in the step 4-7 to give a compound represented byformula (XXXXII) which is a compound represented by formula (Ia) whereinR^(5ax) and R^(4″eq) represent a hydrogen atom and R^(5eq) and R^(4″ax)represent a hydroxyl.

Specifically, this step can be carried out in the same manner as in step2-10 in scheme 5.

(5) Production of Compounds of which the 5-Position is Axial and the4″-Position is Equatorial: First Production Process

In the first production process according to the present invention, acompound represented by formula (Ia), wherein both R^(5eq) and R^(4″ax)represent a hydrogen atom and both R^(5ax) and R^(4″eq) representhydroxyl, can be produced according to the following scheme 10 (step 5-1to step 5-6) and scheme 11 (step 5-7 to step 5-13).

The production process of compounds represented by formula (Xa), whereinthe steric configuration of the hydroxyl group at 5-position is axial,will be specifically described with reference to step 5-1 to step 5-6 inscheme 10. In scheme 10, the compound represented by formula (Xa),wherein the steric configuration of the hydroxyl group at 5-position isaxial, corresponds to the compound represented by formula (XXXXVIII).

wherein, R^(5ax) represents hydroxyl; and R^(5eq) represents a hydrogenatom.

wherein, R¹, R³, R^(2′), R^(6′), and R^(3″) are as defined above; R⁵represents a protective group for hydroxyl group and is selected from,for example, ester-type protective groups such as acetyl or benzoyl andsulfonyl-type protective groups such as p-toluenesulfonyl,methanesulfonyl, or trifluoromethanesulfonyl; and R² and R^(2″) arepreferably an ester-type protective group.

Step 5-1

Step 5-1 is a step of introducing protective groups into five aminogroups in 2-hydroxygentamicin C1a represented by formula (II).

The protective group introduced is preferably the protective groupexemplified in scheme 2. In this step for producing a compoundrepresented by formula (XXXXIII), tert-butoxycarbonyl is particularlypreferred. The protective group can be introduced under conditionsdescribed in detail in connection with step 1-7 in scheme 2.

Step 5-2

Step 5-2 is a step of introducing protective groups (R² and R^(2″)) intotwo hydroxyl groups in the compound represented by formula (XXXXIII). Anester-type protective group such as acetyl is preferred as theprotective group introduced, and acetyl is selected for the compoundrepresented by formula (XXXXIV).

The acetyl group can be introduced in the same manner as in step 3-4 inscheme 6.

Step 5-3

Step 5-3 is a step of introducing a leaving group into the tertiaryhydroxyl group of the compound represented by formula (XXXXIV) in thepresence of a base to cause elimination and thus to form an unsaturatedbond and, at the same time, introducing a leaving group into thehydroxyl group at 5-position to give a compound represented by formula(XXXXV).

The leaving group to be introduced may be selected from alkylsulfonylgroups such as methanesulfonyl and trifluoromethanesulfonyl, orarylsulfonyl groups such as p-toluenesulfonyl. In the production of thecompound represented by formula (XXXXV), methanesulfonyl is preferred.The reaction is carried out by treating sulfonyl chloride and thecompound represented by formula (XXXXIV) in the presence of a base.Bases usable in the reaction include organic bases such as triethylamineor 4-dimethylaminopyridine. Preferred is 4-dimethylaminopyridine.

Any solvent may be used in the reaction without particular limitation sofar as the solvent is inert to the reaction. Preferred are halidesolvents such as methylene chloride.

Step 5-4

Step 5-4 is a step of inverting the steric configuration at 5-positionof the compound represented by formula (XXXXV) to introduce protectedhydroxyl group and thus to synthesize a compound represented by formula(XXXXVI). An ester group is preferred as the protected hydroxyl group inconsideration of a deprotection step which will be described later. Inthe compound represented by formula (XXXXVI), acetyl is preferred.

This step of introducing acetyl to invert the hydroxyl group at5-position can be carried out in the same manner as in step 4-3 inscheme 8.

Step 5-5

Step 5-5 is a step of removing the three protective groups (R², R^(2″),R⁵) for hydroxyl groups in the compound represented by formula (XXXXVI)to give a compound represented by formula (XXXXVII).

In the compound represented by formula (XXXXVI), the three protectivegroups (R², R^(2″), and R⁵) for hydroxyl groups are acetyl. Thedeprotection thereof can be carried out under the same conditions as instep 3-1 of scheme 6 or under conditions similar to the conditions instep 3-1 of scheme 6.

Step 5-6

Step 5-6 is a step of subjecting the compound represented by formula(XXXXVII) to acid hydrolysis reaction to give a compound represented byformula (XXXXVIII).

This reaction can be carried out by reference to the reaction conditionsof step 1-6 in scheme 2. A solvent, which does not inhibit the reaction,such as methanol, may also be additionally used from the viewpoint ofenhancing the solubility of the compound represented by formula(XXXXVII) to accelerate the reaction.

Step 5-7

Step 5-7 is a step of introducing protective groups into the four aminogroups in the compound represented by formula (XXXXVIII) produced instep 5-6 to give a compound represented by formula (XXXXIX).

The protective group to be introduced is preferably a protective groupexemplified in scheme 2, more preferably p-toluenesulfonyl. Conditionsdescribed in detail in step 1-7 of scheme 2 may be applied to theintroduction of the protective group.

The compound represented formula (Ia), wherein both R^(5eq) and R^(4″ax)represent a hydrogen atom and both R^(5ax) and R^(4″eq) representhydroxyl, can be produced according to scheme 12 (step 5-8 to step5-14). In scheme 12, the compound is represented by formula (XXXXXVI).

Scheme 11 includes the step of reacting the compound represented byformula (XXXXIX) with the compound represented by formula (Xc) (step5-8), the step of removing the protective groups of the resultantcompound and converting the azido group in the compound to amino group(step 5-9 and step 5-10), the step of optionally introducing aprotective groups into functional groups other than the amino group at1-position in the resultant compound (step 5-11 and step 5-12), the stepof reacting the resultant compound with the compound represented byformula (XVII) (step 5-13), and the step of removing the protectivegroups in the resultant compound to give a contemplated compoundrepresented by formula (XXXXXVI) (step 5-14). In scheme 11, the compoundrepresented by formula (Xc) corresponds to the compound represented byformula (VIII) or formula (IX).

wherein R¹, R³, R^(2′), R^(6′), R^(2″), B, E, F, G, X, n, and the stericconfiguration of carbon atom attached with * are as defined above; and,R^(4″) and R^(6″) independently represent protective groups forhydroxyl, which are respectively the same protective groups for hydroxylgroups as defined in scheme 1, and are selected from ester-typeprotective groups such as acetyl and benzoyl.

Step 5-8

Step 5-8 is a step of condensing the compound represented by formula(XXXXIX) produced in scheme 10 with the compound represented by formula(VIII) produced in scheme 1 or the compound represented by formula (IX)to give a compound represented by formula (XXXXX). In formula (VIII) orformula (IX), R^(4″) and R^(6″) which are an ester-type protective groupfor hydroxyl group are preferably acetyl.

The reaction in this step can be achieved under the same conditions usedin step 1-8 in scheme 3 or under conditions similar to the conditionsused in step 1-8 in scheme 3.

Step 5-9

Step 5-9 is a step of removing the two protective groups (R^(4″) andR^(6″)) for hydroxyl groups in the compound represented by formula(XXXXX) produced in step 5-8 to give a compound represented by formula(XXXXXI).

The two protective groups (R^(4″) and R^(6″)) for hydroxyl groups in thecompound represented by formula (XXXXX) are acetyl and can be removed inthe same manner as in step 3-1 in scheme 6.

Step 5-10

Step 5-10 is a step of reducing the azido group in the compoundrepresented by formula (XXXXXI) produced in step 5-9 to amino group andfurther removing the four protective groups (R¹, R³, R^(2′) and R^(6′))for amino groups and the protective group (R^(2″)) for hydroxyl group inone step to give a compound represented by formula (XXXXXII).

This step is the same as step 1-10 in scheme 3, and conditions for Birchreduction in the conditions for step 1-10 can be applied.

Step 5-11

Step 5-11 is a step of selectively introducing protective groups (R^(2′)and R^(6′)) into the amino groups at 2′- and 6′-positions in thecompound represented by formula (XXXXXII) produced in step 5-10 to givea compound represented by formula (XXXXXIII). In formula (XXXXXIII),R^(2′) and R^(6′) represent benzyloxycarbonyl, and the reactionconditions described in step 1-11 in scheme 3 can be applied.

Step 5-12

Step 5-12 is a step of selectively introducing a protective group(R^(3″)) into the amino group at 3″-position of the compound representedby formula (XXXXXIII) to give a compound represented by formula(XXXXXIV). In formula (XXXXXIV), preferably, R^(3″) representstrifluoroacetyl, and the reaction conditions for step 1-12 in scheme 3can be applied to the reaction.

Step 5-13

Step 5-13 is a step of reacting the amino group at the 1-position of thecompound represented by formula (XXXXXIV) with aω-amino-α-hydroxycarboxylic acid derivative represented by formula(XVII) to give a compound represented by formula (XXXXXV), that is, astep of conducting a reaction for peptide bond formation. This step canbe carried out in the same manner as in step 1-13 in scheme 3.

Step 5-14

Step 5-14 is a step of removing all the protective groups in thecompound represented by formula (XXXXXV) to give a compound representedby formula (Ia) wherein both R^(5eq) and R^(4″ax) represent a hydrogenatom and both R^(5ax) and R^(4″eq) represent hydroxyl.

This step can be achieved in the same manner as in step 1-14 in scheme3.

(6) Production of Compound of which 5-Position is Axial and 4″-Positionis Axial: Inversion Reaction of Steric Configuration at 4″-Position

The compound represented by formula (Ia), wherein both R^(5eq) andR^(4″ax) represent a hydrogen atom and both R^(5ax) and R^(4″ax)represent hydroxyl, can be produced according to scheme 12 (step 6-1 to6-8). In scheme 12, this compound is represented by formula (XXXXXXIV).

Scheme 12 includes the step of introducing protective groups into thefunctional groups other than the functional group at 4″-position of thecompound represented by formula (XXXXXVI) produced in scheme 11 (step6-1 to step 6-5), the step of inverting the steric configuration of thehydroxyl group at 4″-position (step 6-6), and the step of removing theprotective groups (step 6-7 and step 6-8).

wherein R³, R^(2′), R^(6′), R^(3″), and E represent a protective groupfor amino group, preferably t-butoxycarbonyl; R², R^(2″), and Grepresent an ester-type protective group such as acetyl and benzoyl,preferably acetyl; R^(4″) represents a group selected from the groupconsisting of ester-type protective groups such as acetyl and benzoyland sulfonyl-type protective groups such as p-toluenesulfonyl,methanesulfonyl and trifluoromethanesulfonyl; R^(6″) represents aprotective group for hydroxyl group which can be removed under acidiccondition such as triphenylmethyl; A represents C1-6 lower alkyl, or twoof A together may form five-membered or eight-membered cyclic alkyl; andn and the steric configuration of carbon atom attached with * are asdefined above.

Step 6-1

Step 6-1 is a step of introducing protective groups into the five aminogroups in the compound represented by formula (XXXXXVI) produced inscheme 11 to give a compound represented by formula (XXXXXVII). Thisstep can be carried out by the method described in detail in step 1-7 inscheme 2.

Step 6-2

Step 6-2 is a step of introducing cyclic acetal, preferablycyclohexylidene acetal, as a protective group into the two hydroxylgroups in the compound represented by formula (XXXXXVII). This step issimilar to step 2-2 in scheme 4 and can be achieved by applying thereaction conditions adopted in step 2-2 in scheme 4.

Step 6-3

Step 6-3 is a step of protecting the three hydroxyl groups in thecompound represented by formula (XXXXXVIII) produced in step 6-2 byester-type protective groups.

Conditions for introducing an ester-type protective group for hydroxylgroup in organic synthesis, for example, the conditions applied in step3-4 in scheme 6, can be applied to this step.

Step 6-4

Step 6-4 is a step of removing the cyclic acetal as the protective groupin the compound represented by formula (XXXXXIX) produced in step 6-3 togive a compound represented by formula (XXXXXX).

The conditions adopted in step 2-4 in scheme 4 can be applied to thisstep.

Step 6-5

Step 6-5 is a step of selectively introducing a protective group intothe primary hydroxyl group in the compound represented by formula(XXXXXX) produced in step 6-4 to give a compound represented by formula(XXXXXXI).

A particularly bulky protective group selected from protective groupsused as protective groups for hydroxyl group in conventional organicsynthesis, specifically, for example, triphenylmethyl, is selected asthe protective group to be introduced. The reaction is carried out byallowing triphenylmethyl chloride or triphenylmethyl bromide chloride toact on the compound represented by formula (XXXXXX) in the presence of abase. Any base may be used without particular limitation so far as thebase is inert to the reaction. Preferred bases are, for example,pyridine and 4-dimethylaminopyridine. Further, in this reaction, anysolvent may be used without particular limitation so far as the solventis inert to the reaction.

Step 6-6

Step 6-6 is a step of inverting the hydroxyl group at 4″-position in thecompound represented by formula (XXXXXXI) produced in step 6-5 to give acompound represented by formula (XXXXXXII).

This step is a step similar to the two steps, step 4-2 and step 4-3, inscheme 8 and can be carried out by applying the reaction conditions.Step 6-6 may also be divided into two steps as in scheme 8.

Step 6-7

Step 6-7 is a step of removing the four ester-type protective groups(R², R^(2″), R^(4″), and G) among the protective groups for hydroxylgroup in the compound represented by formula (XXXXXXII) produced in step6-6 to give a compound represented by formula (XXXXXXIII). This step canbe carried out by applying step 2-9 in scheme 5.

Step 6-8

Step 6-8 is a step of removing all the protective groups in the compoundrepresented by formula (XXXXXXIII) produced in step 6-7 to give acompound represented by formula (XXXXXXIV) which is a compoundrepresented by formula (Ia) wherein both R^(5eq) and R^(4″eq) representa hydrogen atom and both R^(5ax) and R^(4″ax) represent hydroxyl.

In formula (XXXXXXIII), reaction conditions for the deprotection dependupon the selected protective group. For example, when R³, R^(2′),R^(6′), R^(3″), and E represent t-butoxycarbonyl while R^(6″) representstriphenylmethyl, the deprotection can be carried out under acidicconditions, for example, using trifluoroacetic acid.

As described above, according to the reaction shown in scheme 12, in thecompound represented by formula (Ia) produced in the first or secondproduction process according to the present invention, the stericconfiguration of the hydroxyl at the 4″-position can be inverted toaxial. Accordingly, the first or second production process according tothe present invention may comprise introducing a protective group intothe functional group other than the hydroxyl group at the 4″-position ofthe compound represented by formula (Ia), inverting the stericconfiguration of the hydroxyl group at the 4″-position, and removing theprotective group to give a compound represented by formula (Ia) whereinthe steric configuration of the hydroxyl group at the 4″-position hasbeen inverted. The present invention includes this embodiment as well

(7) Production of Compound of which 5-Position is Axial and 4″-Positionis Axial: Production of Synthetic Intermediate of which 5-Position isAxial and 4″-Position is Axial (First Production Process)

In the first production process according to the present invention, thecompound represented by formula (Ia), wherein both R^(5eq) and R^(4″eq)represent a hydrogen atom and both R^(5ax) and R^(4″ax) representhydroxyl, can be produced using a synthetic intermediate producedaccording to scheme 13.

In scheme 13, the compound represented by formula (XXXXXXVII)corresponds to a compound represented by formula (Xb) which is asynthetic intermediate in the first production process according to thepresent invention and of which the steric configuration of the hydroxylgroup at the 5-position and 4″-position is axial.

wherein both R^(5eq) and R^(4″ax) represent a hydrogen atom; and bothR^(5ax) and R^(4″ax) represent hydroxyl.

wherein R¹, R³, R^(2′), and R^(6′) represent protective groups for aminogroups, preferably p-toluenesulfonyl; R^(2″) represents an ether-typeprotective group such as benzyl, preferably benzyl; R^(4″) represents anester-type protective group such as acetyl or benzoyl, preferablyacetyl; R represents an ester-type protective group such as acetyl orbenzoyl, preferably benzoyl; B represents a sulfur atom-containingleaving group such as methylthio, ethylthio, or phenylthio, preferablyphenylthio; and X represents a halogen atom such as chlorine, bromine,or iodine, preferably a bromine atom.

Step 7-1

Step 7-1 is a step of condensing the compound represented by formula(XXXXIX) produced in scheme 10 with the compound represented by formula(XXXVIII) or the compound represented by formula (XXXIX) in scheme 8 togive a compound represented by formula (XXXXXXV). This step can becarried out by the method which has been described in detail in step 2-8in scheme 5.

Step 7-2

Step 7-2 is a step of removing the two ester-type protective groups(R^(4″) and R^(6″)) in the compound represented by formula (XXXXXXV) togive a compound represented by formula (XXXXXXVI). This step is similarto step 5-9 in scheme 11 and can be achieved by applying the reactionconditions adopted in step 5-9 in scheme 11.

Step 7-3

Step 7-3 is a step of removing all the protective groups in the compoundrepresented by formula (XXXXXXVI) produced in step 7-2, reducing theazido group to amino to give a compound represented by formula(XXXXXXVII).

The reaction conditions for the deprotection of the compound representedby formula (XXXXXXVII) vary depending upon the selected protectivegroup. For example, when R¹, R³, R^(2′), and R^(6′) representp-toluenesulfonyl, the deprotection can be carried out, for example, byBirch reduction in which a radical reaction is carried out using liquidammonia and metallic sodium.

It would be apparent to a person having ordinary skill in the art that,after step 7-3, the compound represented by formula (Ia) can be producedby applying step 1-11 to step 1-14 in scheme 3 to the compoundrepresented by formula (XXXXXXVII).

The compounds according to the present invention and the compoundsproduced in the production steps can be purified and isolated byconventional purification operation. The purification and isolation canbe carried out, for example, by liquid separation, distillation,sublimation, precipitation, crystallization, column chromatography onsilica gel in which normal- or reverse-phase silica gel is packed,column chromatography using an ion exchange resin such as AmberliteCG-50, Dowex 50 W×2, or CM-Sephadex C-25, column chromatography usingcellulose or the like, preparative thin-layer chromatography or highperformance liquid chromatography. Alternatively, the compounds producedin the above productions steps can also be properly used in subsequentsteps without the isolation and purification.

Antimicrobial Agent

The compounds represented by formula (Ia) according to the presentinvention or their pharmacologically acceptable salts, or their solvateshave excellent antimicrobial activity against bacteria, which causesinfectious diseases, for example, MRSA, staphylococcus aureus,colibacillus, and Pseudomonas aeruginosa, and are preferably used asantimicrobial agents, more preferably anti-MRSA agents. Thus, accordingto another aspect of the present invention, there is provided use of acompound according to the present invention or its pharmacologicallyacceptable salt or their solvates for the manufacture of anantimicrobial agent. Further, according to still another preferredaspect of the present invention, there is provided use of a compoundaccording to the present invention or its pharmacologically acceptablesalt or their solvates for the manufacture of an anti-MRSA agent.

Pharmaceuticals

The compounds represented by formula (Ia) according to the presentinvention or their pharmacologically acceptable salts, or their solvatesoptionally together with pharmaceutically acceptable additives may beused as pharmaceuticals. Thus, according to a further aspect of thepresent invention, there is provided a composition, especially apharmaceutical composition comprising a compound represented by formula(Ia) or its pharmacologically acceptable salt, or their solvate.Further, according to another aspect of the present invention, there isprovided use of a compound represented by formula (Ia) or itspharmacologically acceptable salt, or their solvate for the manufactureof a pharmaceutical composition. The pharmaceutical compositionaccording to the present invention can be specifically used forpreventing or treating an infectious disease. Preferred infectiousdiseases for which the pharmaceutical composition according to thepresent invention is very effective for the treatment or prevention,include nosocomial infectious diseases and opportunistic infectiousdiseases. More preferred are skin suppurative diseases, tympanitis,sinusitis, conjunctivitis, pneumonia, bronchitis, sepsis, cystitis,pyelonephritis, enteritis (including food poisoning) and the like.

The pharmaceutical composition according to the present invention can beadministered to patients parenterally or orally by administrationroutes, for example, parenteral administration (for example, intravenousadministration, intramuscular administration, subcutaneousadministration, rectal administration, or percutaneous administration),or oral administration depending, for example, upon the type ofpathogenic bacteria and diseases and the nature of the patient. Further,the pharmaceutical composition according to the present invention may beformulated into a suitable dosage form depending upon the administrationroute. Examples of such dosage forms include, for example, injectionsused mainly, for example, for intravenous administration andintramuscular administration; external preparations for parenteraladministration, for example, eye drops, ear drops, nasal drops,ophthalmic ointments, skin mucosa absorbers, dermatologic preparations,inhalants, or suppositories; and preparations for oral administration,for example, capsules, tablets, pills, fine subtilaes, granules,powders, syrups, or troches.

The above preparations can be produced by a conventional method usingadditives, for example, excipients, extenders, binders, wetting agents,disintegrators, surfactants, lubricants, dispersants, buffers,preservatives, solubilizers, antiseptics, corrigents, soothing agents,and stabilizers. Specific examples of nontoxic additives usable herein,for injections, eye drops, ear drops, and nasal drops, include,dissolving agents or solubilizers which can constitute aqueous ordissolution-before-use dosage forms (for example, distilled water forinjection, physiological saline, ethanol, glycerin, propylene glycol,corn oils, and sesame oils), pH adjustors (for example, inorganic acidaddition salts such as trisodium orthophosphate and sodiumhydrogencarbonate, organic acidic salts such as sodium citrate, andorganic basic salts such as L-lysine and L-arginine), tonicity adjustingagents (for example, sodium chloride, glucose, and glycerin), bufferingagents (for example, sodium chloride, benzalkonium chloride, and sodiumcitrate), surfactants (for example, sorbitan monooleate and polysorbate80), dispersing agents (for example, D-mannitol), stabilizers (forexample, antioxidants such as ascorbic acid, sodium sulfite, and sodiumpyrosulfite and chelate agents such as citric acid and tartaric acid);for ophthalmic ointments, skin mucosa absorbers, and dermatologicpreparations, include, for example, preparation components suitable asointments, creams, and patch preparations (for example, whitepetrolatum, macrogol, glycerin, liquid paraffin, and cotton clothes);for liquid inhalants, include, for example, pH adjustors (for example,sodium citrate and sodium hydroxide), tonicity adjusting agents (forexample, sodium chloride, benzalkonium chloride, and sodium citrate),and buffering agents (for example, sodium chloride, benzalkoniumchloride, and sodium citrate); for powdery inhalants, include, forexample, lactose as a carrier; and, for preparations for oraladministration and suppositories, include, for example, excipients (forexample, lactose, D-mannitol, corn starch, and crystalline cellulose),disintegrators (for example, carboxymethylcellulose andcarboxymethylcellulose calcium), binders (for example,hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone), lubricants (for example, magnesium stearate and talc),coating agents (for example, shellac, hydroxypropylmethylcellulose,saccharose, and titanium oxide), plasticizers (for example, glycerin andpolyethylene glycol), and substrates (for example, cacao butter,polyethylene glycol, and hard fat).

Further, when an enhancement in the therapeutic or preventive effect ofinfectious diseases is taken into consideration, the pharmaceuticalcomposition according to the present invention may contain, in additionto the compound according to the present invention, clinically usefulone or more conventional antimicrobial agents (for example, β-lactamantimicrobial agents (for example, carbapenems, cephalosporins,cephamycins, and penicillins), glycopeptide antimicrobial agents,ansamycin antimicrobial agents, aminoglycoside antimicrobial agents,quinolone antimicrobial agents, monobactam antimicrobial agents,macrolide antimicrobial agents, tetracycline antimicrobial agents,chloramphenicol antimicrobial agents, lincomycin antimicrobial agents,streptogramin antimicrobial agents, oxazolidinone antimicrobial agents,fosfomycins, novobiocins, cycloserines, and moenomycins). Alternatively,the pharmaceutical composition together with the above antimicrobialagent may be administered to the living body. Further, when the factthat the pharmaceutical composition according to the present inventioncan expand or improve the effectiveness against gram-negative bacteriaand resistant bacterial against existing antimicrobial agents is takeninto consideration, the pharmaceutical composition according to thepresent invention may contain, for example, a drug discharge pump(efflux pump) inhibitor and an existing antimicrobial agent degradativeenzyme (for example, β-lactamase) inhibitor. Alternatively, thepharmaceutical composition according to the present invention, togetherwith these inhibitors or the like may be administered to the livingbody. Furthermore, when the fact that the pharmaceutical compositionaccording to the present invention can enhance the therapeutic orpreventive effect of infectious diseases is taken into consideration,the pharmaceutical composition according to the present invention can beused in combination with compounds not having any antimicrobial activity(for example, medicaments for treating complication). The presentinvention includes this embodiment.

As described above, the compound represented by formula (Ia) accordingto the present invention or its pharmacologically acceptable salt, ortheir solvate can be advantageously utilized as an antimicrobial agentor a pharmaceutical in the prevention or treatment of infectiousdiseases. Thus, according to another aspect of the present invention,there is provided a method for preventing or treating an infectiousdisease, comprising administering the compound represented by formula(Ia) or its pharmacologically acceptable salt, or their solvate to ananimal including a human. Animals as candidates for the prevention ortreatment are preferably mammals, more preferably humans. The dose ofthe compound represented by formula (Ia) or its pharmacologicallyacceptable salt, or their solvate may be appropriately determined by aperson having ordinary skill in the art, for example, in considerationof dose regimen, the type of pathogenic bacteria, and the age, sex, andweight of patients, and disease severity of patients. In particular, thedose per day and the number of doses per day may, if necessary, beappropriately varied.

EXAMPLES

The present invention is further illustrated by the following Examplesthat are not intended as a limitation of the invention.

In the following description, all temperatures are expressed in Celsiusdegree.

¹H-NMR denotes a proton nuclear magnetic resonance spectral method, and¹³C-NMR denotes a carbon nuclear magnetic resonance spectral method.Further, chemical shifts obtained therefrom are expressed in shifts(ppm) from tetramethylsilane (TMS) to a lower magnetic filed side.

MS denotes a mass spectral method, and the results

obtained by an electron spray ionization method (ESI), an atmosphericpressure ionization method (API), a fast atom bombardment method (FAB),and a high-performance liquid chromatography-mass analysis method (LCMS)are expressed in m/z (mass/charge).

Rf values in thin-layer chromatography are values measured with a silicagel plate of ART5715 manufactured by Merck, and a developing solvent,which gave the Rf values, are described within the parentheses.

In the following structural formulae, Bn represents benzyl, Ac acetyl,Ph phenyl, Ts p-toluenesulfonyl, and Cbz benzyloxycarbonyl.

Example 1 Production of 2-hydroxyarbekacin

Production Step 1-1 Methyl3-azido-3-deoxy-4,6-O-isopropylidene-D-glucopyranoside

Methyl 3-azido-3-deoxy-D-glucopyranoside (32.7 g) synthesized accordingto the description of the method of C. B. Barlow et al. (J. Chem. Soc.,Abstracts pp. 3870-3871, (June), (1965)) was dissolved in 330 mL ofN,N-dimethylformamide. 2,2-Dimethoxypropane (26.8 mL) and 1.92 g ofp-toluenesulfonic acid were added to the solution, and the mixture wasstirred at 50° C. Three hr after the start of the stirring, 26.8 mL of2,2-dimethoxypropane was further added to the reaction solution; after5.5 hr, 2.10 g of p-toluenesulfonic acid was further added to thereaction solution; after 6.5 hr, 17.9 mL of 2,2-dimethoxypropane wasfurther added to the reaction solution; and after 24 hr, 16.2 mL oftriethylamine was added under ice cooling to the reaction solution, andthe mixture was concentrated to dryness with a vacuum pump. Chloroform(1.5 L) was added to the residue. The solution was washed twice with 500mL of a saturated aqueous sodium bicarbonate solution and was furtherwashed twice with 500 mL of saturated brine, was dried over Glauber'ssalt, and was concentrated to dryness to give the title compound (amixture of an α form and a β form, 36.3 g, yield 96%) as a brown syrup.

ESIMS: m/z 282 [M+Na]⁺

α Form

¹H-NMR (CDCl₃): δ 4.74 (d, 1H, J=4 Hz), 3.88 (dd, 1H, J=5, 10 Hz), 3.72(t, 1H, J=10, 10 Hz), 3.65 (dddd 1H, J=5, 9, 10 Hz), 3.61 (t, 1H, J=10,10 Hz), 3.55 (dt, 1H, J=4, 10, 10 Hz), 3.45 (s, 3H), 3.47 (t, 1H, J=10,10 Hz), 2.35 (d, 1H, J=10 Hz), 1.51 (s, 3H), 1.44 (s, 3H).

β Form

¹H-NMR (CDCl₃): δ 4.27 (d, 1H, J=8 Hz), 3.95 (dd, 1H, J=5, 10 Hz), 3.78(t, 1H, J=10, 10 Hz), 3.64 (m, 1H), 3.56 (t, 1H, J=10, 10 Hz), 3.55 (s,3H), 3.50 (t, 1H, J=10, 10 Hz), 3.37 (dd, 1H, J=8, 10 Hz), 2.67 (br. s,1H), 1.51 (s, 3H), 1.44 (s, 3H).

Production Step 1-2 Methyl3-azido-2-O-benzyl-3-deoxy-4,6-O-isopropylidene-D-glucopyranoside

The above compound (46.0 g) product in production step 1-1 was dissolvedin 690 mL of N,N-dimethylformamide. Sodium hydride 11.4 g (60% oilsuspension) was added to the solution with stirring under ice coolingand a nitrogen atmosphere, and the mixture was further stirred under icecooling and a nitrogen atmosphere for 30 min. Benzyl bromide (27.4 mL)was added to the reaction solution, and the mixture was stirred under anitrogen atmosphere at room temperature for 1.5 hr. The reactionsolution was then ice cooled and was adjusted to pH 4 to 5 by theaddition of a 50% aqueous acetic acid solution. The reaction mixture wasthen concentrated to dryness, and 2.0 L of chloroform was added thereto.The solution was washed twice with 500 mL of a saturated aqueous sodiumbicarbonate solution and was washed once with 500 mL of water. Thewashed solution was dried over Glauber's salt and was concentrated todryness to give 73.4 g of a crude product. The crude product waspurified by column chromatography on silica gel (hexane:ethylacetate=7:1 to 3:1) using 350 g of a neutral silica gel to give thetitle compound (a mixture of an α form and a β form: 55.1 g, yield 89%).

ESIMS: m/z 372 [M+Na]⁺

α Form

¹H-NMR (CDCl₃): δ 7.30-7.40 (m, 5H), 4.70-4.90 (ABq, 2H, Jgem=12 Hz),4.52 (d, 1H, J=4 Hz), 3.85 (dd, J=2, 10 Hz), 3.82 (t, 1H, J=10, 10 Hz),3.66 (t, 1H, J=10, 10 Hz), 3.65 (t, 1H, J=10, 10 Hz), 3.36 (s, 3H), 3.39(dt, 1H, J=2, 10, 10 Hz), 3.37 (dd, 1H, J=4, 10 Hz), 1.47 (s, 3H), 1.43(s, 3H).

β Form

¹H-NMR (CDCl₃): δ 7.30-7.40 (m, 5H), 4.71-4.88 (ABq, 2H, Jgem=12 Hz),4.38 (d, 1H, J=8 Hz), 3.93 (dd, J=5, 10 Hz), 3.75 (t, 1H, J=10, 10 Hz),3.56 (s, 3H), 3.51 (t, 1H, J=10, 10 Hz), 3.45 (t, 1H, J=10, 10 Hz), 3.27(dd, 1H, J=8, 10 Hz), 3.26 (dt, 1H, J=5, 10, 10 Hz), 1.49 (s, 3H), 1.44(s, 3H).

Production Step 1-3 Methyl 3-azido-2-O-benzyl-3-deoxy-D-glucopyranoside

An 80% aqueous acetic acid solution (186 mL) was added to 37.3 g of thecompound produced in production step 1-2. A reaction was allowed toproceed at 80° C., and, 30 min after the start of the reaction, thereaction solution was cooled to room temperature and was concentrated todryness to give 32.9 g of the title compound (a mixture of an α form anda β form) at a quantitative yield.

ESIMS: m/z 332 [M+Na]⁺

α Form

¹H-NMR (CDCl₃): δ 7.30-7.45 (m, 5H), 4.63-4.80 (ABq, 2H, Jgem=12 Hz),4.57 (d, 1H, J=3.5 Hz), 3.78-3.85 (m, 2H), 3.63 (dt, 1H, J=4, 4, 10 Hz),3.44 (t, 1H, J=10, 10 Hz), 3.40 (dd, 1H, J=3.5, 10 Hz), 3.38 (t, 1H,J=10, 10 Hz), 3.37 (s, 3H), 2.44 (d, 1H, J=3.5 Hz), 1.82 (dd, 1H, J=6,7.5 Hz).

β Form

¹H-NMR (CDCl₃): δ 7.30-7.45 (m, 5H), 4.71-4.92 (ABq, 2H, Jgem=12 Hz),4.39 (d, 1H, J=8 Hz), 3.91 (m, 1H), 3.78-3.85 (m, 2H), 3.58 (s, 3H),3.45 (t, 1H, J=10, 10 Hz), 3.39 (t, 1H, J=10, 10 Hz), 3.27 (dd, 1H, J=8,10 Hz), 2.50 (d, 1H, J=2.5 Hz), 1.97 (dd, 1H, J=6, 7.5 Hz).

Production Step 1-41,4,6-Tri-O-acetyl-3-azido-2-O-benzyl-3-deoxy-D-glucopyranose

Acetic acid-acetic anhydride-concentrated sulfuric acid (50:50:1) (164mL) was added to and dissolved in 32.8 g of the compound produced inproduction step 1-3 with an ultrasonic cleaner, and a reaction wasallowed to proceed at room temperature for 5 hr. The reaction solutionwas dropped with vigorous stirring to 1.7 L of an ice cooled 10% aqueoussodium acetate solution. Chloroform (20 mL) was added four times to thereaction solution, and mixture was vigorously stirred under ice coolingfor 20 min. The reaction solution was returned to room temperature andwas further vigorously stirred for one hr. The reaction solution wasthen extracted with 2.5 L of chloroform (once with 900 mL and twice with800 mL). The chloroform layer was washed once with 500 mL of saturatedbrine, twice with 500 mL of a saturated aqueous sodium bicarbonatesolution, and once with 500 mL of saturated brine. The chloroform layerwas further dried over Glauber's salt and was concentrated to dryness togive 48.4 g of a crude product. The crude product thus obtained waspurified by column chromatography on silica gel (250 g) (hexane:ethylacetate=5:1) to give the title compound (a mixture of an α form and a βform: 41.3 g, yield 92%) as a light yellow syrup.

ESIMS: m/z 444 [M+Na]⁺

α Form

¹H-NMR (CDCl₃): δ 7.30-7.40 (m, 5H), 6.32 (d, 1H, J=4 Hz), 4.90 (t, 1H,J=10, 10 Hz), 4.62-4.71 (ABq, 2H, Jgem=12 Hz), 4.22 (dd, 1H, J=5, 13Hz), 4.01 (dd, 1H, J=2.5, 10 Hz), 3.98 (m, 1H), 3.88 (t, 1H, J=10, 10Hz), 3.58 (dd, 1H, J=4, 10 Hz), 2.06, 2.12, 2.17 (each s, each 3H).

β Form

¹H-NMR (CDCl₃): δ 7.30-7.40 (m, 5H), 5.62 (d, 1H, J=9 Hz), 4.91 (t, 1H,J=10, 10 Hz), 4.72-4.82 (ABq, 2H, Jgem=12 Hz), 4.25 (dd, 1H, J=5, 13Hz), 3.99 (dd, 1H, J=2.5, 13 Hz), 3.74 (ddd, 1H, J=2.5, 5, 10 Hz), 3.66(t, 1H, J=10, 10 Hz), 3.59 (dd, 1H, J=9, 10 Hz), 2.07, 2.12, 2.22 (eachs, each 3H).

Production Step 1-5a4,6-Di-O-acetyl-3-azido-2-O-benzyl-1-bromo-3-deoxy-α-D-glucopyranose

The compound (347 mg) synthesized in production step 1-4 was dissolvedin a mixed solution composed of 6.2 mL of methylene chloride and 0.69 mLof ethyl acetate. Titanium tetrabromide (605 mg) was added to the mixedsolution with stirring under ice cooling, and the mixture was stirred atroom temperature for 14.5 hr. The reaction solution was ice cooled, and30 mL of ice cooled methylene chloride was added thereto. The solutionwas washed eight times with 15 mL of ice cooled water until the waterlayer had pH 7. The solution was then dried over Glauber's salt underice cooling and was concentrated to dryness to give the title compound(352 mg, yield: 97%) as a light yellow syrup.

¹H-NMR (CDCl₃): δ 7.20-7.45 (m, 5H), 6.32 (d, 1H, J=3.5 Hz), 4.92 (t,1H, J=10, 10 Hz), 4.68-4.74 (ABq, 2H, Jgem=12 Hz), 4.27 (dd, 1H, J=4.5,12.5 Hz), 4.17 (ddd, 1H, J=2.5, 4.5, 10 Hz), 4.03 (dd, 1H, J=2.5, 12.5Hz), 3.98 (t, 1H, J=10, 10 Hz), 3.43 (dd, 1H, J=3.5, 10 Hz), 2.13 (s,3H), 2.06 (s, 3H).

Production Step 1-5b4,6-Di-O-acetyl-3-azido-2-O-benzyl-3-deoxy-1-thiophenyl-α-D-glucopyranose

The compound (19.9 g) produced in production step 1-4 was dissolved in200 mL of methylene chloride. Trimethylsilylthiophenol (26.8 mL) and11.0 mL of trimethylsilyltriflate were added to the solution, and themixture was refluxed. The reaction mixture was ice cooled 41 hr afterthe start of the reflux. Further, ice cooled chloroform (1.8 L) wasadded thereto. The reaction mixture was then washed three times with 1 Lof an ice cooled 5% aqueous NaOH solution and twice with 1 L of icecooled water. The reaction mixture was then dried over Glauber's salt,was concentrated to dryness to give a crude product (22.1 g). The crudeproduct was then dissolved in 20 mL of ethyl acetate. Hexane (120 mL)was further added to the solution for recrystallization. The resultantcrystal was washed with ice cooled ethyl acetate:hexane (1:9) to givethe title compound (17.7 g, yield 79%). Further, the mother liquor andthe wash liquid for the crystal were combined and were concentrated todryness to give the title compound (4.3 g).

ESIMS: m/z 494 [M+Na]⁺

¹H-NMR (CDCl₃): δ 7.20-7.48 (m, 10H), 5.59 (d, 1H, J=5 Hz), 4.83 (t, 1H,J=10, 10 Hz), 4.68-4.78 (ABq, 2H, Jgem=12 Hz), 4.46 (ddd, 1H, J=2.5, 5,10 Hz), 4.22 (dd, 1H, J=5, 12 Hz), 3.96 (t, 1H, J=2.5, 12 Hz), 3.86 (t,1H, J=10, 10 Hz), 3.79 (dd, 1H, J=5, 10 Hz), 2.13 (s, 3H), 1.99 (s, 3H).

Production Step 1-6 3′,4′-Dideoxy-2-hydroxyneamine

Process A: 2-Hydroxygentamicin C1a (18.0 g) represented by formula (II)was produced according to the description of Japanese Patent Laid-OpenNo. 108041/1976 and was purified. Next, 3 M HCl (360 mL) was added tothe resultant 2-hydroxygentamicin C1a, and the mixture was refluxed for1.5 hr. The reaction solution was returned to room temperature, was thenice cooled, was adjusted to pH about 6.8 by the addition of 4 M NaOH,was diluted with 1.8 L of water, and was added into an Amberlite CG-50(previously equilibrated with 0.005 M aqueous ammonia) 500 mL column.This column was washed with 1 L of 0.005 M aqueous ammonia. Elution wascarried out with 2 L of 0.1 M aqueous ammonia and 2 L of 0.3 M aqueousammonia. Fractions containing the title compound were combined and wereconcentrated to dryness to give 6.1 g of the title compound. Fractionscontaining impurities were again purified by the Amberlite CG-50 columnto give 0.6 g of the title compound. Thus, 6.7 g in total of the titlecompound (monocarbonate, yield 72%) was obtained.

Process B: 2-Hydroxygentamicin C1a (600 g) represented by formula (II)was produced and was purified according to the description of JapanesePatent Laid-Open No. 108041/1976. Next, 2-hydroxygentamicin C1a (300 g,483 mmol as 2.5 carbonate) was dissolved in 3 N HCl (3 L), and thesolution was stirred at 95° C. for 70 min. After standing to cool, thesolution was ice cooled to 10° C. and was neutralized and adjusted to pH6.88 by the addition of 5 N NaOH. Further, the same reaction was againcarried out using 2-hydroxygentamicin C1a (300 g, 483 mmol as 2.5carbonate). The resultant two reaction mixtures were combined and werepurified with Amberlite CG-50 (NH₄ type; equilibrated with 0.005 M NH₃,10 L). Elution solvent: 0.1 M→0.2 M→0.3 M NH₃. The purified product waslyophilized to give the title compound (335.9 g; 781 mmol asdicarbonate; yield 81%).

ESIMS: m/z 329 [M+Na]⁺

¹H-NMR (26% ND₃-D₂O: δ 5.09 (d, 1H, J=3.5 Hz), 3.82 (m, 1H), 3.56 (t,1H, J=10, 10 Hz), 3.30 (t, 1H, J=10, 10 Hz), 3.35 (t, 1H, J=10, 10 Hz),3.08 (t, 1H, J=10, 10 Hz), 2.82 (dt, 1H, J=3.5, 3.5, 12 Hz), 2.78 (t,1H, J=10, 10 Hz), 2.58-2.68 (m, 2H), 2.63 (t, 1H, J=10, 10 Hz),1.67-1.79 (m, 2H), 1.60 (dq, 1H, J=4, 12, 12, 12 Hz), 1.36 (dq, 1H, J=4,12, 12, 12 Hz).

Production Step 1-73′,4′-Dideoxy-2-hydroxyl-1,3,2′,6′-tetra-N-tosylneamine

The compound (6.7 g) produced in production step 1-6 was dissolved in 67mL of water. Sodium carbonate (11.6 g) was added to the solution, and134 mL of dioxane was further added. p-Toluenesulfonyl chloride (20.8 g)was added to the mixture under ice cooling, and the mixture wasvigorously stirred for 15 min under ice cooling. The solution wasreturned to room temperature and was vigorously stirred overnight.Dioxane (134 mL) was then added to the reaction solution; after 42.5 hr,3.9 g of sodium carbonate was added to the reaction solution; after 88hr, 3.5 g of p-toluenesulfonyl chloride was added to the reactionsolution; and after 112.5 hr, 8.5 mL of concentrated aqueous ammonia wasadded to the reaction solution, and the mixture was stirred for 30 min.The reaction mixture was concentrated to dryness. Ethyl acetate (700 mL)was added to the residue, and the mixture was washed twice with 300 mLof water, was dried over Glauber's salt, and was then concentrated todryness. The crude product was purified by column chromatography onsilica gel (chloroform:methanol=20:1) on 250 g of silica gel to give3.47 g of the title compound. A fraction containing impurities was alsosubjected to column chromatography on silica gel to give 7.05 g of thetitle compound. As a result, 10.52 g (total amount; yield 63%) of thetitle compound was produced.

ESIMS: m/z 945 [M+Na]³⁰

¹H-NMR (Pyridine-d5): δ 9.20 (d, 1H, J=7 Hz), 9.15 (d, 1H, J=8 Hz), 8.80(d, 1H, J=8 Hz), 8.46 (t, 1H, J=6, 6 Hz), 7.90-8.18 (m, 8H), 7.00-7.15(m, 8H), 5.97 (d, 1H, J=3 Hz), 5.09 (m, 1H), 4.14 (t, 1H, J=10, 10 Hz),4.07 (t, 1H, J=10, 10 Hz), 3.91 (t, 1H, J=10, 10 Hz), 3.79-3.86 (m, 2H),3.74 (t, 1H, J=10, 10 Hz), 3.73 (m, 1H), 3.30-3.47 (m, 2H), 2.40 (dq,1H, J=5, 12, 12, 12 Hz), 2.17 (s, 6H), 2.16 (s, 3H), 2.11 (s, 3H), 1.77(m, 1H), 1.58-1.72 (m, 2H).

Production Step 1-84″,6″-Di-O-acetyl-3″-azido-2″-O-benzyl-3″-deoxy-2-hydroxyl-1,3,2′,6′-tetra-N-tosyldibekacin

Process A: A solution of 352 mg of the compound produced in productionstep 1-5a in 7.3 mL of 1,2-dichloroethane was added to 363 mg of thecompound produced in production step 1-7. Drierite (1070 mg) was addedthereto, and the mixture was stirred at room temperature for 30 min.Mercuric cyanide (397 mg) was added to the reaction solution, and themixture was stirred under light shielding at 60° C. until the compoundproduced in production step 1-5a disappeared when the reaction wastraced by TLC. The reaction solution was returned to room temperatureand was filtered through Celite. The insolubles were washed with 73 mLof chloroform. The filtrate and the wash liquid were combined, werewashed three times with 36 mL of a saturated aqueous sodium bicarbonatesolution, three times with 36 mL of a 10% aqueous sodium iodide solutionand twice with 36 mL of water in that order, were dried over a Glauber'ssalt, and were concentrated to dryness. The residue was purified bycolumn chromatography on silica gel (chloroform:ethyl acetate=5:2) on 36g of silica gel to give the title compound (190 mg, yield 37.5%).

Process B: The compound (10.52 g) produced in production step 1-7 andthe compound (9.67 g) produced in production step 1-5b were dissolved in210 mL of methylene chloride. A molecular sieves 4 A powder (31.6 g) wasadded to the solution, and the mixture was stirred at room temperaturefor 30 min. Next, a solution of 5.54 g of N-iodosuccinimide and 0.55 mLof trifluoromethanesulfonic acid in 9.45 mL of methylene chloride wasadded to the reaction solution with stirring at −20° C. under lightshielding. Further, the mixture was stirred at −20° C. for 30 min underlight shielding. Next, 3.4 mL of triethylamine was added to the reactionsolution, and the mixture was filtered through Celite, and theinsolubles were washed with 1.8 L of chloroform. Next, the filtrate andthe wash liquid were combined, were washed twice with 1 L of a saturatedaqueous sodium bicarbonate solution, twice with 1 L of a 10% aqueoussodium thiosulfate solution, and twice with 500 mL of water in thatorder, were dried over a Glauber's salt, and were concentrated todryness to give 20.2 g of a crude product. Next, the crude product waspurified by column chromatography on 500 g of silica gel(chloroform:ethyl acetate=5:2) to give the title compound (5.4 g, yield37%).

Rf value 0.68 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1306 [M+Na]⁺

¹H-NMR (Pyridine-d5): δ 9.22 (d, 1H, J=7.5 Hz), 8.82 (d, 1H, J=8.5 Hz),8.73 (t, 1H, J=6, 6 Hz), 8.25 (d, 1H, J=8.5 Hz), 7.8-8.1 (m, 10H),7.0-7.45 (m, 11H), 6.75 (d, 1H, J=3 Hz), 6.09 (d, 1H, J=2 Hz), 5.51(ABq, 1H, Jgem=11 Hz), 5.23 (t, 1H, J=10, 10 Hz), 5.03 (m, 1H), 4.96 (m,1H), 4.93 (ABq, 1H, Jgem=11 Hz), 4.41 (t, 1H, J=10, 10 Hz), 4.38 (dd,1H, J=4, 12 Hz), 4.23 (m, 1H), 4.07-4.21 (m, 5H), 3.83 (dd, 1H, J=3, 10Hz), 3.78 (t, 1H, J=9, 9 Hz), 3.72 (m, 1H), 3.27 (m, 1H), 3.17 (m, 1H),2.35 (m, 1H), 2.21 (s, 3H), 2.20 (s, 6H), 2.19 (s, 3H), 2.03 (s, 3H),2.01 (s, 3H), 1.65 (m, 1H), 1.45-1.60 (m, 2H).

Production Step 1-93″-Azido-2″-O-benzyl-3″-deoxy-2-hydroxyl-1,3,2′,6′-tetra-N-tosyldibekacin

A 0.1% solution (118 mL) of sodium methoxide in methanol was added to5.91 g of the compound produced in production step 1-8, and the mixturewas allowed to react at room temperature. A 28% solution (1.74 mL) ofsodium methoxide in methanol was added to the reaction solution 50 minafter the start of the reaction. After 1.5 hr, the reaction solution wasneutralized and adjusted to pH 6 to 7 with Dowex 50W×2 (H⁺ form, 200-400mesh, substituted with MeOH). Next, the resin was removed from thereaction solution by filtration, and the insolubles were washed fivetimes or more with methanol. The filtrate and the wash liquid werecombined and were concentrated to dryness to give 5.2 g of the crudetitle compound. This compound as such was used in the next reaction.

Rf value 0.39 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1222 [M+Na]⁺

Production Step 1-10 2-Hydroxydibekacin

Liquid ammonia (about 10 mL) was stored at −50° C. in an egg-plant typeflask containing 45.7 mg of the compound produced in production step1-9. Next, 70 mg of metallic sodium was added to the egg-plant typeflask at −50° C., and the mixture was vigorously stirred with a glassstirrer bar for 2 hr. Methanol was slowly added to the egg-plant typeflask until the color of radicals disappeared. The mixture was returnedto room temperature to evaporate ammonia and was finally concentrated todryness with an evaporator. Water (3 mL) was added to the egg-plant typeflask, and the contents of the flask were adjusted to pH 4 to 5 withDowex 50W×2 (H⁺ form). The contents of the flask together with the resinwere added to a column paced with 2 mL of the same resin. The column waswashed with 20 mL of water, was eluted with 1 M aqueous ammonia.Ninhydrin positive fractions were combined and were concentrated todryness to give 20.1 mg of a crude product. This was brought to anaqueous solution (10 mL) which was then added to a CM-Sephadex C-25column (equilibrated with 0.005 M aqueous ammonia, 10 mL). The columnwas washed with water (10 mL). Next, elution was carried out whilesuccessively changing the concentration of aqueous ammonia from 0.05M(50 mL) to 0.2 M (100 mL, 2 mL cut), and the corresponding fraction wasconcentrated to dryness to give 16.4 mg of the title compound(monocarbonate-monohydrate, yield 79%).

Rf value 0.35 (1-butanol:ethanol:chloroform:17% aqueousammonia=4:7:2:7).

ESIMS: m/z 490 [M+Na]⁺

¹H-NMR (26% ND₃-D₂O): δ 5.12 (d, 1H, J=3.5 Hz), 5.01 (d, 1H, J=4 Hz),3.88 (m, 1H), 3.83 (m, 1H), 3.75 (dd, 1H, J=2.5, 12 Hz), 3.68 (dd, 1H,J=5, 12 Hz), 3.66 (t, 1H, J=10, 10 Hz), 3.46 (dd, 1H, J=4, 10 Hz), 3.35(t, 1H, J=10, 10 Hz), 3.28 (t, 1H, J=10, 10 Hz), 3.27 (t, 1H, J=10, 10Hz), 3.10 (t, 1H, J=10, 10 Hz), 2.99 (t, 1H, J=10, 10 Hz), 2.83 (t, 1H,J=10, 10 Hz), 2.80 (m, 1H), 2.79 (t, 1H, J=10, 10 Hz), 2.66 (dd, 1H,J=4.5, 13 Hz), 2.62 (dd, 1H, J=7, 13 Hz), 1.68-1.77 (m, 2H), 1.60 (m,1H), 1.37 (m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 102.62, 101.41, 85.70, 84.53, 75.36, 74.00,73.67, 72.89, 71.83, 70.35, 61.35, 57.67, 56.81, 55.65, 51.35, 46.60,28.97, 27.45.

Production Step 1-11 2′,6′-Di-N-benzyloxycarbonyl-2-hydroxydibekacin

Nickel acetate tetrahydrate (1136 mg) was added to the compound (625 mg;as calculated as monocarbonate.monohydrate) produced in production step1-10. Methanol (25 mL) was added to thereto, and the mixture was treatedwith an ultrasonic cleaner to give a homogeneous solution.N-Benzyloxycarbonyloxysuccinimide (626 mg) was added little by little tothe solution under ice cooling over a period of about 2 min. The mixturewas stirred under ice cooling for one hr, was returned to roomtemperature, and was further stirred for 2.5 hr. Next, 57 mg ofN-benzyloxycarbonyloxysuccinimide was added to the reaction solutionunder ice cooling, was returned to room temperature, and was furtherstirred for 2 hr. The reaction solution was concentrated to dryness.Concentrated aqueous ammonia (30 mL) saturated with sodium chloride wasadded to the residue, and the mixture was extracted three times with 20mL of 1-butanol. The butanol layer thus obtained was concentrated todryness. N,N-Dimethylformamide was added to 1423 mg of the residue andwas filtered through Celite. The substance on the Celite was washed withN,N-dimethylformamide (20 mL×1, 10 mL×4). The filtrate and the washliquid were concentrated to dryness to give 1222 mg of a crude product.

Rf value 0.60 (1-butanol:ethanol:chloroform:17% aqueous ammonia=4:7:2:7)

ESIMS: m/z 758 [M+Na]⁺

¹H-NMR (Pyridine-d5): δ 8.35 (br. s, 1H), 7.90 (d, 1H, J=7.5 Hz),7.15-7.55 (m, 10H), 5.62 (d, 1H, J=3 Hz), 5.41 (d, 1H, J=3 Hz), 5.33 (s,2H), 5.30 (ABq, 1H, Jgem=12 Hz), 5.14 (ABq, 1H, Jgem=12 Hz), 4.64 (m,1H), 4.44 (dd, 1H, J=2, 12 Hz), 4.42 (m, 1H), 4.28 (dd, 1H, J=5, 12 Hz),4.24 (t, 1H, J=10, 10 Hz), 4.20 (dd, 1H, J=3, 10 Hz), 4.05 (m, 1H), 4.04(t, 1H, J=10, 10 Hz), 3.90 (t, 1H, J=10, 10 Hz), 3.85 (t, 1H, J=10, 10Hz), 3.68 (t, 1H, J=10, 10 Hz), 3.60 (t, 1H, J=10, 10 Hz), 3.52 (m, 1H),3.39 (m, 1H), 3.29 (t, 1H, J=10, 10 Hz), 3.11 (t, 1H, J=10, 10 Hz), 2.05(m, 1H), 1.89 (dq, 1H, J=3.5, 13, 13, 13 Hz), 1.64 (m, 1H), 1.43 (q, 1H,J=13, 13, 13 Hz).

Production Step 1-122′,6′-Di-N-benzyloxycarbonyl-2-hydroxy-3″-N-trifluoroacetyldibekacin

The crude product (1222 mg) produced in production step 1-11 wasdissolved in 17 mL of anhydrous N,N-dimethylformamide. Ethyltrifluoroacetate (0.15 mL) was added to the solution with stirring underice cooling. The mixture was returned to room temperature and wasstirred for 8 hr. The reaction solution was concentrated to dryness togive 1416 mg of the product.

Rf value 0.42 (lower layer part of a solution of chloroform:methanol: 15M aqueous ammonia (concentrated aqueous ammonia)=1:1:1 was used)

¹H-NMR (Pyridine-d5): δ 10.65 (d, 1H, J=9 Hz), 8.35 (br. s, 1H), 7.89(d, 1H, J=8.5 Hz), 7.10-7.55 (m, 10H), 5.70 (d, 1H, J=3 Hz), 5.41 (d,1H, J=3 Hz), 5.33 (s, 2H), 5.30 (ABq, 1H, Jgem=12 Hz), 5.26 (ABq, 1H,Jgem=12 Hz), 5.18 (m, 1H), 4.77 (m, 1H), 4.58 (t, 1H, J=10, 10 Hz), 4.50(dd, 1H, J=3, 10 Hz), 4.31-4.46 (m, 3H), 4.18 (t, 1H, J=10, 10 Hz), 4.04(m, 1H), 3.96 (t, 1H, J=10, 10 Hz), 3.82 (t, 1H, J=10, 10 Hz), 3.71 (t,1H, J=10, 10 Hz), 3.51 (m, 1H), 3.40 (m, 1H), 3.31 (t, 1H, J=10, 10 Hz),3.10 (t, 1H, J=10, 10 Hz), 2.05 (m, 1H), 1.89 (m, 1H), 1.64 (m, 1H),1.43 (m, 1H).

Production Step 1-131-N-(4-Benzyloxycarbonylamino-2-(S)-hydroxybutyryl)-2′,6′-di-N-benzyloxycarbonyl-2-hydroxy-3″-N-trifluoroacetyldibekacin

The product (438 mg) produced in production step 1-12 was dissolved in6.2 mL of tetrahydrofuran. A solution (2 mL) of 149 mg of an(S)-4-N-benzyloxycarbonylamino-2-hydroxybutyric acid succinimide estersynthesized according to a report of Kawaguchi et al. (Journal ofAntibiotics, Vol. 25, pp. 695 to 708 (1972)) in tetrahydrofuran wasadded dropwise to the solution with stirring under ice cooling over aperiod of 2 min, and the mixture was returned to room temperature andwas stirred. A solution (2 mL) of 149 mg of anN-benzyloxycarbonyl-4-amino-2-(S)-hydroxybutyrylic acid succinimideester in tetrahydrofuran was added to the reaction solution withstirring under ice cooling 19 hr after the start of stirring, and themixture was returned to room temperature and was stirred. After 20.5 hr,the reaction solution was concentrated to dryness.

Ethyl acetate (70 mL) was added to the residue, and the mixture waswashed twice with 14 mL of a saturated aqueous sodium bicarbonatesolution and twice with 14 mL of water and was concentrated to drynessto give 571 mg of the reaction mixture.

Rf value 0.59 (lower part of a solution of chloroform:methanol:15 Maqueous ammonia (concentrated aqueous ammonia)=1:1:1 was used)

Production Step 1-14 2-Hydroxyarbekacin

Tetrahydrofuran (22.8 mL) and 17.8 mL of 3.5 M aqueous ammonia wereadded to 571 mg of the reaction mixture produced in production step1-13, and the mixture was stirred at room temperature for 20 hr. Thereaction solution was concentrated to dryness. Tetrahydrofuran-aceticacid-water (4:1:1) (22 mL) was added to 624 mg of the residue. Further,12 drops of a suspension of palladium black in water was added, and themixture was stirred at the atmospheric pressure for 6 hr while blowing ahydrogen gas into the system. Next, palladium black was removed from thereaction solution by filtration. The removed palladium black was washedwith water, and the filtrate and the wash liquid were combined and wereconcentrated to dryness. 2 M aqueous ammonia (15 mL) was added to theresidue, and the mixture was allowed to stand at room temperatureovernight. The insolubles were removed by filtration through a cottonstopper, followed by concentration to dryness to give 467 mg of a crudeproduct. The crude product was dissolved in water to give 50 mL of anaqueous solution, and the aqueous solution added to a CM-Sephadex C-25column (equilibrated with 0.005 M aqueous ammonia, 50 mL). The columnwas washed with 100 mL of 0.005 M aqueous ammonia, and elution wascarried out with aqueous ammonia with 0.05 M (250 mL) to 0.5 M (500 mL)and further 0.75 M (500 mL). The corresponding fractions wereconcentrated to give 39.1 mg of the title compound: 2-hydroxyarbekacin.

¹H-NMR (26% ND₃-D₂O): δ 5.13 (d, 1H, J=3.5 Hz), 5.03 (d, 1H, J=4 Hz),4.16 (dd, 1H, J=4, 9.5 Hz), 3.98 (m, 1H), 3.83-3.90 (m, 2H), 3.65-3.77(m, 4H), 3.37 (t, 1H, J=10, 10 Hz), 3.34 (t, 1H, J=10, 10 Hz), 3.32 (dd,1H, J=4, 10 Hz), 3.25 (t, 1H, J=10, 10 Hz), 2.97 (t, 1H, J=10, 10 Hz),2.86 (t, 1H, J=10, 10 Hz), 2.82 (m, 1H), 2.70-2.78 (m, 2H), 2.65 (dd,1H, J=5, 13 Hz), 2.62 (dd, 1H, J=7, 13 Hz), 1.86-1.96 (m, 1H), 1.69-1.80(m, 3H), 1.61 (dq, 1H, J=4, 13, 13, 13 Hz), 1.37 (dq, 1H, J=4, 13, 13,13 Hz).

¹³C-NMR (26% ND₃-D₂O): δ 177.84, 102.09, 98.99, 83.65, 78.17, 75.41,72.87, 72.23, 71.71, 71.28, 70.45, 69.81, 60.79, 56.60, 56.15, 54.88,50.71, 46.02, 38.26, 37.23, 28.39, 26.94.

Example 2 2-Hydroxyarbekacin 2.5 sulfate trihydrate

The compound (2-hydroxyarbekacin; 126.8 mg) represented by formula (I)produced in production step 1-14 of Example 1 was brought to an aqueoussolution. The aqueous solution was adjusted to pH 7.0 by the addition of0.1 M sulfuric acid and was lyophilized to give 147.3 mg of 2.5 sulfate(trihydrate) of the title compound.

Calcd. for C₂₂H₄₄N₆O₁₁.2.5H₂SO₄.3H₂O. C, 30.45; H, 6.39; N, 9.67.

Found, C, 30.41; H, 6.45; N, 9.46.

Example 3 Production of 2-hydroxyarbekacin

Production step 3-11,3,2′,6′-Tetra-N-benzyloxycarbonyl-3,4′-dideoxy-2-hydroxyneamine

Process A: The compound (16.5 g, 38.3 mmol: calculated as dicarbonate)produced in production step 1-6 was dissolved in water (83 ml).Dimethoxyethane (165 ml) was added to the solution, and the mixture wasthen stirred. N-Benzyloxycarbonyloxysuccinimide (66.8 g, 268 mmol) wasadded thereto, triethylamine (56.0 ml, 402 mmol) was then added, and themixture was stirred overnight (heat up to about 45° C. was generated).Ethyl acetate (490 ml) and water (83 mL) were added to the reactionsolution followed by separation. The organic layer was washed with asaturated aqueous sodium hydrogencarbonate solution (247 mL) and a 10%aqueous sodium thiosulfate solution (247 mL) in that order. The organiclayer was dried over anhydrous magnesium sulfate, was filtered, and wasthen concentrated under the reduced pressure. Methanol (133 ml) wasadded to the residue (as a foam), and the mixture was stirred on a waterbath heated to 60° C. As a result, crystals were precipitated. Themixture was stirred at room temperature overnight (slurry washing). Theprecipitated crystals were collected by filtration and were dried underthe reduced pressure at 40° C. overnight to give the title compound(yield 33.8 g, quantitative).

Process B: The compound (303 g, 704 mmol as dicarbonate) produced inproduction step 1-6 was dissolved in water (1.5 L), and1,2-dimethoxyethane (3.0 L) was added to the solution.N-Benzyloxycarbonyloxysuccinimide (1229 g, 4.93 mol, 7.0 eq.) was addedthereto, and triethylamine (1013 mL, 7.40 mol, 10.5 eq.) was then added,and the mixture was stirred overnight. Ethyl acetate (9.1 L) and water(1.5 L) were added to the reaction solution, followed by separation. Thewater layer was again extracted with ethylacetate:1,2-dimethoxyethane=3:1 (1.8 L). The organic layers werecombined and were washed once with a 5% aqueous sodium hydrogencarbonatesolution (4.6 L) and once with a 10% aqueous sodium thiosulfate solution(4.6 L). The organic layer was dried over anhydrous magnesium sulfate,and the solvent was then removed by distillation under the reducedpressure. Methanol (2.4 L) was added to the residue, and the mixture wasstirred at room temperature overnight. The precipitated crystal wasfiltered to give the title compound (619.1 g, 734 mmol, yield 104%).

APIMS: m/z 843 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 7.73 (d, 1H, J=6.9 Hz), 7.12-7.56 (m, 22H),6.82-6.95 (m, 1H), 5.67 (d, 1H, J=2.9 Hz), 5.12-5.31 (m, 8H), 4.26-4.40(m, 1H), 3.90-4.30 (m, 7H), 3.35-3.50 (m, 2H), 1.85-2.01 (m, 2H),1.43-1.65 (m, 2H).

Production Step 3-21,3,2′,6′-Tetra-N-benzyloxycarbonyl-5,6-O-cyclohexylidene-3′,4′-dideoxy-2-hydroxyneamine

Process A: The compound (2.0 g, 2.4 mmol) produced in production step3-1 was dissolved in dimethoxyethane (40 mL). 1,1-Dimethoxycyclohexane(0.54 mL, 3.56 mmol) and pyridinium p-toluenesulfonate (PPTS) (0.060 g,0.24 mmol) were added to the solution, and the mixture was stirred on anoil bath at 110° C. for 2 hr with an apparatus comprising a droppingfunnel containing molecular sieves 5 A 1/16 (30 g, 40 mL) and a Dimrothcondenser provided on the dropping funnel (internal temperature 85° C.).Ethyl acetate (40 mL) and a saturated aqueous sodium hydrogencarbonatesolution (40 mL) were added to the reaction solution, followed byseparation. The organic layer was washed with a saturated aqueous NaClsolution and was dried over anhydrous magnesium sulfate and was thenconcentrated under the reduced pressure. The residue was purified bycolumn chromatography on silica gel (hexane:ethyl acetate=2:1 to 1:3,chloroform:methanol=9:1) to give the title compound (1.75 g, 1.89 mmol,yield 80%).

Process B: The compound (150 g, 178 mmol) produced in production step3-1 was suspended in 1,2-dimethoxyethane (3.0 L).1,1-Dimethoxycyclohexane (38.5 g, 267 mmol, 1.5 eq.) and pyridiniump-toluenesulfonate (4.47 g, 17.8 mmol, 0.1 eq.) were added, and themixture was heated and was refluxed while passing through 1.5 kg ofmolecular sieves 5 A ( 1/16) for 4 hr. The reaction mixture was dilutedwith ethyl acetate (3 L) and was washed once with a 5% aqueous sodiumhydrogencarbonate solution (4.6 L) and once with a 10% aqueous sodiumthiosulfate solution (3 L). The organic layer was dried over anhydrousmagnesium sulfate, and the solvent was then removed by distillationunder the reduced pressure. The same reaction was further carried outthree more times. The residues were combined and were purified bychromatography on silica gel to give the title compound. Developingsolvent=hexane:ethyl acetate=2:1→1:1→1:2→1:3. (557.93 g, 604 mmol, yield85%).

APIMS: m/z 923 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 7.96 (d, 1H, J=6.2 Hz), 7.68 (d, 1H, J=6.8 Hz),7.22-7.57 (m, 20H), 6.75-7.05 (m, 2H), 5.57 (s, 1H), 5.12-5.35 (m, 8H),3.96-4.45 (m, 6H), 3.92 (t, 1H, J=10, 10 Hz), 3.79 (t, 1H, J=10, 10 Hz),3.30-3.47 (m, 2H), 1.77-2.00 (m, 2H), 1.45-1.70 (m, 10H), 1.16-1.35 (m,2H).

Production Step 3-32-Benzyloxy-1,3,2′,6′-tetra-N-benzyloxycarbonyl-5,6-O-cyclohexylidene-3′,4′-dideoxyneamine

Process A: Tetrahydrofuran (99 mL) was added to the compound (4.970 g,5.38 mmol) produced in production step 3-2. A 60% sodium hydride (0.26g, 6.50 mmol) dispersion in paraffin liquid and benzyl bromide (1.15 mL,9.67 mmol) were added at an internal temperature of 4° C., and themixture was stirred at an internal temperature of 7 to 8° C. for 21 hr.A saturated aqueous ammonium chloride solution (10 mL) was added theretoat an internal temperature of 4° C., and the mixture was adjusted to pH7, followed by separation with 100 mL of ethyl acetate and 50 mL ofwater. The organic layer was dried over magnesium sulfate, and thesolvent was removed by distillation under the reduced pressure. Theresidue was purified by column chromatography on silica gel(toluene:ethyl acetate=3:1) to give the title compound (4.492 g, 4.43mmol, yield 83%).

Process B: The compound (396 g, 429 mmol) produced in production step3-2 was dissolved in a mixed solution composed of tetrahydrofuran (8 L)and N,N-dimethylformamide (158 mL), and the solution was cooled to 5° C.Sodium hydride (dispersion in paraffin liquid, 60%; 20.6 g, 515 mmol,1.2 eq.) and benzyl bromide (91.8 mL, 772 mmol, 1.8 eq.) were added tothe solution, and the mixture was stirred at the same temperature for 6hr. A 10% aqueous ammonium chloride solution (8 L) was added thereto tostop the reaction, and the mixture was extracted with ethyl acetate (8L). The organic layer was washed once with 20% brine (8 L) and once witha 10% aqueous sodium thiosulfate solution (8 L) and was dried overanhydrous magnesium sulfate. The solvent was then removed bydistillation under the reduced pressure. The residue was purified bychromatography on silica gel to give the title compound. Developingsolvent=hexane:ethyl acetate=1:1→1:3 (320.24 g, 316 mmol, yield 74%).

APIMS: m/z 1014 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.08-8.16 (m, 1H), 7.88-7.96 (m, 1H), 7.10-7.48(m, 25H), 6.70-6.95 (m, 2H), 5.53 (s, 1H), 5.15-5.46 (m, 8H), 4.91 (AB,2H, Jgem=11 Hz), 4.40-4.51 (m, 1H), 4.20-4.35 (m, 2H), 3.93-4.16 (m,4H), 3.72 (t, 1H, J=10, 10 Hz), 3.32-3.50 (m, 2H), 1.77-2.00 (m, 2H),1.45-1.72 (m, 10H), 1.15-1.35 (m, 2H).

Production Step 3-42-Benzyloxy-1,3,2,6′-tetra-N-benzyloxycarbonyl-3,4′-dideoxyneamine

Process A: The compound (22.75 g, 22.5 mmol) produced in production step3-3 was dissolved in a mixed solution composed of chloroform (460 mL)and methanol (46 mL), and the solution was cooled to 7° C. 90%trifluoroacetic acid (46 mL) was added to the cooled solution, and themixture was stirred at room temperature for 2 hr. The solvent wasremoved by distillation under the reduced pressure. Methanol (460 mL)was added to the residue, and the mixture was subjected to slurrywashing for 2 hr, followed by filtration to give the title compound(19.47 g, 0.9 mmol, yield 93%).

Process B: The compound (376 g, 371 mmol) produced in production step3-3 was dissolved in a mixed solution composed of chloroform (7.5 L) andmethanol (750 mL), and the mixture was cooled to 10° C. 90%trifluoroacetic acid (750 mL) was added to the cooled solution, and themixture was stirred at 20° C. for 3 hr 20 min. The solvent was removedby distillation under the reduced pressure. Methanol (7.5 L) was addedto the residue, and the slurry was stirred overnight and was filtered togive the title compound (313 g, 335 mmol, yield 90%).

APIMS: m/z 933 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 7.86 (d, 1H, J=6.6 Hz), 7.62-7.70 (m, 1H),7.10-7.55 (m, 26H), 6.80-6.95 (m, 1H), 5.64 (d, 1H, J=2.9 Hz), 5.13-5.37(m, 8H), 4.91 (ABq, 2H, Jgem=12 Hz), 4.29-4.37 (m, 3H), 3.90-4.17 (m,3H), 4.02 (dd, 1H, J=9.1, 10 Hz), 3.94 (dd, 1H, J=8.8, 9.0 Hz),3.92-3.52 (m, 2H), 1.86-2.04 (m, 2H), 1.47-1.66 (m, 2H).

Production Step 3-52-Benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-1,6-N,O-carbonyl-3,4′-dideoxyneamine

Process A: The compound (19.291 g, 20.7 mmol) produced in productionstep 3-4 was dissolved in N,N-dimethylformamide (390 mL), and thesolution was cooled to 3° C. Sodium hydride (60%, in oil; 4.968 g, 0.124mol) was added to the cooled solution, and the mixture was stirred atthe same temperature for 1.5 hr. The reaction mixture was adjusted to pHabout 7 under ice cooling by the addition of a saturated aqueousammonium chloride solution (400 mL). Ethyl acetate (800 mL) and asaturated aqueous ammonium chloride solution (400 mL) were addedthereto, and the mixture was stirred, followed by separation. Theorganic layer was washed twice with a 5% aqueous ammonium chloridesolution (800 mL) and was then dried over magnesium sulfate. The solventwas removed by distillation under the reduced pressure. Methanol (400mL) was added to the residue. A seed crystal was added thereto, and acontemplated product was crystallized with gentle stirring. The crystalswere collected by filtration to give the title compound. The motherliquor was concentrated under the reduced pressure, and the residue wassubjected to slurry washing with methanol/diisopropyl ether (1/1; 100mL) to further give the title compound (15.70 g, 19.0 mmol, yield 92%).

Process B: The compound (293 g, 314 mmol) produced in production step3-4 was cooled in N,N-dimethylformamide (5.9 L), and the solution wascooled to 3° C. Sodium hydride (dispersion in paraffin liquid, 60%; 75.4g, 1.88 mol, 6.0 eq.) was added to the cooled solution, and the mixturewas stirred at the same temperature for one hr. The reaction mixture waspoured into a two-layer solution of a 20% aqueous ammonium chloridesolution (12 L) and ethyl acetate (5 L) with vigorous stirring under icecooling. Further, ethyl acetate (7 L) was added thereto, and the mixturewas stirred, followed by separation. The organic layer was washed twicewith a 5% aqueous ammonium chloride solution (12 L) and was dried overanhydrous magnesium sulfate. The solvent was removed by distillationunder the reduced pressure. Methanol (5.9 L) was added to the residue,and the mixture slurry was stirred overnight and was then filtered togive the title compound (248 g, 301 mmol, yield 95%).

APIMS: m/z 825 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.80 (s, 1H), 8.67 (s, 1H), 8.17-8.24 (m, 1H),7.75-7.53 (m, 1H), 7.12-7.53 (m, 20H), 6.93-7.10 (m, 1H), 5.22 (d, 1H,J=3.1 Hz), 5.11-5.39 (m, 6H), 4.88 (ABq, 2H, Jgem=12 Hz), 3.95-4.38 (m,7H), 3.73 (t, 1H, J=10, 10 Hz), 3.38-3.53 (m, 2H), 1.77-1.95 (m, 2H),1.45-1.72 (m, 2H).

Production Step 3-62-Benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxyneamine

Process A: The compound (822 mg, 0.997 mmol) produced in production step3-5 was dissolved in a mixed liquid composed of 1,4-dioxane (25 mL) andwater (25 mL). Sodium carbonate (127 mg, 1.20 mmol) was added to thesolution, and the mixture was stirred at 80° C. for 5 hr. The reactionmixture was cooled and was extracted once with chloroform (50 mL) andonce with a mixed liquid composed of chloroform (25 mL) and methanol (25mL). The extract was dried over magnesium sulfate. The solvent wasremoved by distillation under the reduced pressure. The residue wasdissolved in ethyl acetate (16 mL). A seed crystal was added to thesolution, and a contemplated product was crystallized with gentlestirring, followed by filtration to give compound 7. The mother liquorwas concentrated under the reduced pressure, and the residue wassubjected to slurry washing with ethyl acetate/diisopropyl ether (1/1; 4mL) to further give the title compound (617 mg, 0.772 mmol, yield 77%).

Process B: The compound (228 g, 276 mmol) produced in production step3-5 was dissolved in a mixed liquid composed of 1,4-dioxane (6.8 L) andwater (6.8 L). Sodium carbonate (29.30 g, 276 mmol, 1.0 eq.) was addedthe solution, and the mixture was stirred at 80° C. for 6 hr. Thereaction mixture was cooled, and sodium chloride (1.37 kg) was dissolvedtherein, and the mixture was extracted with chloroform (13.7 L). Theextract was dried over anhydrous magnesium sulfate. The solvent was thenremoved by distillation under the reduced pressure to give the titlecompound (157.87 g, 198 mmol, yield 71%).

FABMS: m/z 799 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 7.10-7.60 (m, 22H), 6.65-6.90 (m, 1H), 5.66 (d,1H, J=2.9 Hz), 5.34 (s, 2H), 5.24 (ABq, 2H, Jgem=13 Hz), 5.21 (ABq, 2H,Jgem=13 Hz), 4.97 (ABq, 2H, Jgem=11 Hz), 3.64-4.36 (m, 5H), 3.89 (t, 1H,J=9.0, 9.0 Hz), 3.54 (t, 1H, J=10, 10 Hz), 3.39-3.50 (m, 2H), 3.07 (t,1H, J=10, 10 Hz), 1.87-2.02 (m, 2H), 1.45-1.65 (m, 2H).

Production Step 3-71-N—[(S)-4-Benzyloxycarbonylamino-2-benzyloxybutyryl]-2-benzyloxy-3,2,6′-tri-N-benzyloxycarbonyl-3,4′-dideoxyneamine

Process A: (S)-2-Benzyloxy-4-benzyloxycarbonylamino-butyric acid (9.065g, 26.4 mmol) synthesized in Reference Example 1 was dissolved intetrahydrofuran, and the solution was cooled to 2° C.N-Hydroxysuccinimide (3.038 g, 26.4 mmol) and dicyclohexylcarbodiimide(5.447 g, 26.4 mmol) were added to the solution, and the mixture wasstirred at room temperature for 2 hr. The insolubles were filtered togive an active ester solution. The compound (10.552 g, 13.2 mmol)produced in production step 3-6 was dissolved in tetrahydrofuran (210mL). Triethylamine (3.7 mL, 27 mmol) and the active ester solution wereadded to the solution, and the mixture was stirred at 50° C. for 3.5 hr.The reaction mixture was diluted with chloroform (800 mL), washed with asaturated sodium hydrogencarbonate solution (800 mL) and was then driedover magnesium sulfate. The solvent was removed by distillation underthe reduced pressure. The residue was purified by column chromatographyon silica gel (chloroform:ethylacetate=1:1→chloroform:methanol=30:1→20:1) to give the title compound(11.352 g, 10.1 mmol, yield 76%).

Process B: (S)-2-Benzyloxy-4-benzyloxycarbonylamino-butyric acid (101.78g, 296 mmol, 1.5 eq.) synthesized in Reference Example 1 was dissolvedin tetrahydrofuran (2.04 L), and the solution was cooled to 3.4° C.N-Hydroxysuccinimide (37.53 g, 326 mmol, 1.65 eq.) anddicyclohexylcarbodiimide (67.28 g, 326 mmol, 1.65 eq.) were added to thecooled solution, and the mixture was stirred at 25° C. for 3 hr. Theinsolubles were filtered to give an active ester solution. The compound(157.87 g, 198 mmol) produced in production step 3-6 was dissolved intetrahydrofuran (3.16 L). Triethylamine (41.34 mL, 296 mmol, 1.5 eq.)and the active ester solution were added to the solution, and themixture was stirred at 53° C. for 4.5 hr. The stirred mixture was thendiluted with chloroform (12.63 L), and the diluted solution was washedwith a 5% aqueous sodium hydrogencarbonate solution (12.63 L) and wasthen dried over anhydrous magnesium sulfate. The solvent was removed bydistillation under the reduced pressure. The residue was purified bycolumn chromatography on silica gel to give the title compound.Developingsolvent=chloroform:acetone=3.5:1→3:1→chloroform:methanol=30:1→20:1.(158.45 g, 140 mmol, yield 71%).

LCMS: m/z 1124 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.15 (d, 1H, J=8.3 Hz), 7.60-7.72 (m, 1H),7.10-7.55 (m, 32H), 6.83-7.00 (m, 1H), 5.65 (d, 1H, J=2.9 Hz), 5.12-5.36(m, 8H), 4.98 (ABq, 2H, Jgem=11, 11 Hz), 4.60 (ABq, 2H, Jgem=12, 12 Hz),3.90-4.49 (m, 8H), 4.08 (t, 1H, J=9.8, 9.8 Hz), 3.38-3.70 (m, 4H),2.20-2.36 (m, 2H), 1.85-2.01 (m, 2H), 1.45-1.65 (m, 2H).

Production Step 3-84″,6″-Di-O-acetyl-3″-azido-2″,2′″-di-O-benzyl-1,3,2′,6′-tetra-N-benzyloxycarbonyl-3″-deoxy-2-hydroxylarbekacin

The compound (11.249 g, 10.0 mmol) produced in production step 3-7 and4,6-di-O-acetyl-3-azido-2-O-benzyl-1,3-dideoxy-1-phenylthio-α-D-glucopyranose(9.440 g, 20.0 mmol), which had been dried under the reduced pressurefor 2 hr, were dissolved in methylene chloride (225 mL). Molecularsieves 4 A (powder, 33.7 g), which had been dried under the reducedpressure for 2 hr, was added to the solution, and the mixture wasstirred at room temperature under an argon atmosphere for one hr. Thereaction vessel was brought to a light shielded state and was cooled to−20° C. N-Iodosuccinimide (10.811 g, 48.1 mmol) andtrifluoromethanesulfonic acid (178 mL, 2.01 mmol) were added thereto,and the mixture was stirred at room temperature for 2 hr.N-Iodosuccinimide (5.405 g, 24.0 mmol) and trifluoromethanesulfonic acid(88 mL, 0.99 mmol) were added, and the mixture was stirred at roomtemperature for additional two hr. Triethylamine (431 mL, 3.09 mmol) wasadded under ice cooling to stop the reaction. The insolubles werefiltered, and the organic layer was washed once with a saturated aqueoussodium hydrogencarbonate solution (500 mL) and twice with a 10% aqueoussodium thiosulfate solution and was dried over magnesium sulfate. Thesolvent was removed by distillation under the reduced pressure. Theresidue was purified by column chromatography on silica gel(chloroform:acetone=10:1→:/1→chloroform:methanol=10:1) to give the titlecompound (9.388 g, 6.32 mmol, yield 63%). The compound (3.16 g, 2.81mmol) produced in production step 3-7 remaining unreacted was recovered.

LCMS: m/z 1485 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.30 (d, 1H, J=8.5 Hz), 7.75-7.83 (m, 1H),7.20-7.67 (m, 36H), 6.92-7.05 (m, 1H), 6.75-6.90 (m, 1H), 5.92 (d, 1H,J=3.4 Hz), 5.65 (d, 1H, J=2.9 Hz), 4.94-5.35 (m, 12H), 4.62-4.76 (m,4H), 4.35-4.50 (m, 3H), 3.92-4.35 (m, 9H), 3.71 (dd, 1H, J=3.4, 10 Hz),3.34-3.62 (m, 4H), 2.18-2.43 (m, 2H), 2.00 (s, 3H), 1.99 (s, 3H),1.80-1.93 (m, 2H), 1.43-1.65 (m, 2H).

Production Step 3-93″-Azido-2″,2′″-di-O-benzyl-1,3,2′,6′-tetra-N-benzyloxycarbonyl-3″-deoxy-2-hydroxylarbekacin

The compound (8.819 g, 5.94 mmol) produced in production step 3-8 wasdissolved in a mixed liquid composed of chloroform (180 mL) and methanol(90 mL). 0.5 M sodium methoxide (a methanol solution, 3.6 mL, 1.8 mmol)was added to the solution, and the mixture was stirred at roomtemperature for 2 hr. Acetic acid (0.14 mL, 2.4 mmol) was added thereto,and the mixture was washed with a saturated aqueous sodium carbonatesolution (250 mL). The water layer was again extracted with chloroform(200 mL). The combined organic layers were dried over magnesium sulfate,and the solvent was removed by distillation under the reduced pressure.The residue was purified by column chromatography on silica gel(chloroform:methanol=30:1) to give the title compound (7.263 g, 5.18mmol, yield 87%).

LCMS: m/z 1401 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.11 (d, 1H, J=8.3 Hz), 7.50-7.62 (m, 1H),7.10-7.58 (m, 35H), 6.96-7.10 (m, 1H), 6.80-6.96 (m, 1H), 6.14-6.23 (m,1H), 5.50-5.65 (m, 3H), 5.12-5.33 (m, 8H), 4.91 (ABq, 2H, Jgem=11, 11Hz), 4.83 (ABq, 2H, Jgem=12, 12 Hz), 4.58 (ABq, 2H, Jgem=12, 12 Hz),3.85-4.51 (m, 14H), 3.37-3.64 (m, 5H), 2.20-2.42 (m, 2H), 1.80-1.95 (m,2H) 1.45-1.65 (m, 2H).

Production Step 3-10 2-Hydroxyarbekacin

The compound (154 mg, 0.110 mmol) produced in production step 3-9 wasdissolved in 3 mL of a solution of 1,4-dioxane:water: 1 N hydrochloricacid=40:19:1. The solution was adjusted to pH 1.53, a palladium blackpowder (154 mg) was added thereto, and the mixture was vigorouslystirred under a hydrogen atmosphere. The pH value of the reaction systemreached 8.50 three hr after the start of vigorous stirring. The reactionmixture was adjusted to pH 1.68 by the addition of 1 N hydrochloric acid(500 μl) and was vigorously stirred under a H₂ atmosphere for 15 hr. Thepalladium black powder was removed by filtration through a cotton, andthe catalyst was washed with water. The filtrate and the wash liquidwere combined and were concentrated to dryness. The concentrate wasdissolved in water to give a 10-mL solution which was then purified by aBio Rex 70 column (equilibrated with 0.005 M aqueous ammonia) to givethe title compound (51.1 mg, 0.079 mmol, yield 72%).

LCMS: m/z 569 [M+H]⁺

¹H-NMR (26% ND₃-D₂O): δ5.13 (d, 1H, J=3.5 Hz), 5.03 (d, 1H, J=4 Hz),4.16 (dd, 1H, J=4, 9.5 Hz), 3.98 (m, 1H), 3.83-3.90 (m, 2H), 3.65-3.77(m, 4H), 3.37 (t, 1H, J=10, 10 Hz), 3.34 (t, 1H, J=10, 10 Hz), 3.32 (dd,1H, J=4, 10 Hz), 3.25 (t, 1H, J=10, 10 Hz), 2.97 (t, 1H, J=10, 10 Hz),2.86 (t, 1H, J=10, 10 Hz), 2.82 (m, 1H), 2.70-2.78 (m, 2H), 2.65 (dd,1H, J=5, 13 Hz), 2.62 (dd, 1H, J=7, 13 Hz), 1.86-1.96 (m, 1H), 1.69-1.80(m, 3H), 1.61 (dq, 1H, J=4, 13, 13, 13 Hz), 1.37 (dq, 1H, J=4, 13, 13,13 Hz).

Example 4 Production of 2-hydroxyarbekacin

Production Step 4-13-Azido-2-O-benzyl-3-deoxy-1-thiophenyl-α-D-glucopyranose

Process A: A 0.13% sodium methoxide/methanol solution (40.5 mL) and 13.5mL of chloroform were added to the compound (2.67 g) of production step1-5b, and the mixture was allowed to react at room temperature for 1.5hr. The reaction mixture was neutralized (pH 6 to 7) by the addition ofDowex 50 W×2 (H⁺ form, substituted by methanol). The resin was removedby filtration followed by washing with methanol (4 mL×5). The filtrateand the wash liquid were combined and were concentrated to dryness togive a crude product (2.193 g, quantitative).

Process B: The compound (26.0 g) produced in production step 1-5b wassuspended in 260 mL of methanol, and 33.3 mL of a 0.5 M sodiummethoxide/methanol solution was added. The mixture was allowed to reactat room temperature for 30 min, and 1 mL of acetic acid was addeddropwise. The reaction solution was concentrated to dryness to give acrude product which as such was used in a next reaction.

ESIMS: m/z 388 [M+H]⁺

¹H-NMR (CDCl₃): δ 7.28-7.47 (m, 10H), 5.56 (d, 1H, J=4.6 Hz), 4.68-4.79(ABq, 2H, Jgem=11.7, 12.3 Hz), 4.21 (dt, 1H, J=3.9, 9.7 Hz), 3.70-3.81(m, 4H), 3.45 (t, 1H, J=9.3, 9.5 Hz).

Production Step 4-22-O-Benzyl-3-benzyloxycarbonylamino-1,3-dideoxy-1-phenylthio-α-D-glucopyranose

Process A: The compound (2.19 g) produced by process A in productionstep 4-1 was dissolved in 44 mL (20 v/w) of anhydrous tetrahydrofuran.Triphenylphosphine (7.43 g, 5 folds by mol) was added to the solution,and the mixture was allowed to react at room temperature. Water (0.71mL, 7 folds by mol), N-benzyloxycarbonyloxysuccinimide (1.83 g, 1.3folds by mol), and triethylamine (2.37 mL, 3 folds by mol) were addedthereto 18.5 hr after the start of the reaction, and the mixture wasstirred at room temperature. The reaction mixture was concentrated todryness 2.5 hr after the start of the stirring at room temperature togive 12.6 g of a crude product.

Process B: The compound (about 22.0 g) produced by process B inproduction step 4-1 was dissolved in a mixed liquid (550 mL, 10 v/mol)of tetrahydrofuran/water (1:1). Triphenylphosphine (36.1 g, 2.5 folds bymol) was added to the solution, and the mixture was allowed to react atroom temperature. N-Benzyloxycarbonyloxysuccinimide (17.84 g, 1.3 foldsby mol) and 10 mL (1.3 folds by mol) of triethylamine were added thereto2 hr after the start of the reaction, and the mixture was stirred atroom temperature. The reaction mixture was concentrated to dryness 2.5hr after the start of the stirring and was washed with ethylacetate/hexane to give 23.7 g of a crude product (yield in three steps87%).

APIMS: m/z 496 [M+H]⁺

¹H-NMR (DMSO): δ 7.52 (dt, 2H, J=1.5, 7.0 Hz), 7.23-7.36 (m, 13H), 5.78(d, 1H, J=4.9 Hz), 5.18 (d, 1H, J=6.8 Hz), 4.97-5.12 (ABq, 2H,Jgem=12.7, 12.9 Hz), 4.48-4.71 (ABq, 2H, Jgem=12.2 Hz), 4.55 (t, 1H,J=5.8 Hz), 3.91 (m, 1H), 3.71 (dd, 1H, J=5.1, 10.7 Hz), 3.53 (m, 2H).

Production Step 4-34,6-Di-O-acetyl-2-O-benzyl-3-benzyloxycarbonylamino-1,3-dideoxy-1-phenylthio-α-D-glucopyranose

Process A: The crude product (12.61 g) produced by process A inproduction step 4-2 was rendered anhydrous and was dissolved in 56 mL ofanhydrous pyridine. Acetic anhydride (5.3 mL, 10 folds by mol) was addedto the solution under ice cooling, and the mixture was allowed to reactat room temperature for 16 hr. Methanol (4.5 mL) (2 folds by mol ascompared with acetic anhydride) was added thereto, and the mixture wasallowed to stand at room temperature for 30 min and was concentrated todryness (azeotropic distillation with toluene: three times). Further,330 mL of chloroform was added to the residue, and the mixture waswashed with a saturated aqueous sodium bicarbonate solution (160 mL×3),a 5% aqueous KHSO₄ solution (160 mL×3), and distilled water (160 mL×1)in that order, was dried over a Glauber's salt, and was concentrated todryness to give 13.3 g of a crude product. The crude product waspurified by column chromatography on silica gel and was thencrystallized from chloroform/hexane to give 3.19 g of a contemplatedproduct as a crystal (yield 97%).

Process B: A part (4.93 g) of a crude product produced by process B inproduction step 4-2 was rendered anhydrous and was dissolved in 50 mL(10 v/w) of pyridine. Acetic anhydride (25 mL, 5 v/w) was added to thesolution, and the mixture was allowed to react at room temperature for 3hr. Methanol (25 mL, 5 v/w) was added thereto under ice cooling, and themixture was allowed to stand at room temperature for 30 min. Next, 150mL of chloroform was added, and the mixture was washed with water (150mL×1), 2.5 N hydrochloric acid (150 mL×1), 0.5 N hydrochloric acid (150mL×1), and a saturated aqueous sodium bicarbonate solution (150 mL×1) inthat order, was dried over magnesium sulfate, was concentrated todryness, and was crystallized from ethyl acetate/hexane to give 5.39 gof a contemplated product (yield 94%).

FABMS: m/z 580 [M+M]⁺

¹H-NMR (CDCl₃): δ 7.46-7.49 (m, 2H), 7.26-7.36 (m, 13H), 5.67 (d, 1H,J=5.4 Hz), 5.12 (bs, 2H), 4.89 (t, 1H, J=9.9, 10.3 Hz), 4.74-4.50 (ABq,2H, Jgem=12.2 Hz), 4.50-4.56 (m, 2H), 4.26 (dd, 1H, J=5.4, 12.2 Hz),4.07 (dd, 1H, J=9.7, 10.3 Hz), 3.96 (dd, 1H, J=2.2, 12.2 Hz), 3.77 (bs,1H), 1.98 (s, 3H), 1.94 (s, 3H).

Production Step 4-44′,6″-Di-O-acetyl-2″,2′″-di-O-benzyl-2-hydroxyl-3,2′,6′,3″-tetra-N-benzyloxycarbonylarbekacin

Process A: The compound (2.401 g, 2.14 mmol) produced in production step3-7 and 4,6-di-O-acetyl-2-O-benzyl-3-benzyloxycarbonylamino-1,3-di deoxy-1-phenylthio-α-D-glucopyranose (1.490 g, 2.57 mmol), which had beendried under the reduced pressure for 2 hr, were dissolved in methylenechloride (48 mL). Molecular sieves 4 A (powder, 7.20 g) dried under thereduced pressure for 2 hr was added to the solution, and the mixture wasstirred under an argon atmosphere at room temperature for one hr. Thereaction vessel was brought to a light shielded state and was cooled to−20° C. N-Iodosuccinimide (1.390 g, 6.18 mmol) andtrifluoromethanesulfonic acid (38 mL, 0.43 mmol) were added thereto, andthe mixture was stirred at room temperature for 2 hr. N-Iodosuccinimide(0.700 g, 3.09 mmol) and trifluoromethanesulfonic acid (19 mL, 0.22mmol) were added to the reaction mixture, and the mixture was stirred atroom temperature for additional 2 hr. Triethylamine (90 mL, 0.65 mmol)was added under ice cooling to stop the reaction. The insolubles werefiltered, and the organic layer was washed once with a saturated aqueoussodium hydrogencarbonate solution (120 mL) and twice with a 10% aqueoussodium thiosulfate solution, and was dried over magnesium sulfate. Thesolvent was removed by distillation under the reduced pressure, and theresidue was purified by column chromatography on silica gel(chloroform:acetone=10:1→7:1→chloroform:methanol=10:1) to give the titlecompound (2.350 g, 1.47 mmol, yield 69%).

Process B: Dichloromethane (1.4 L) was added to the compound (70.0 g,62.3 mmol) produced in production step 3-7 and4,6-di-O-acetyl-2-O-benzyl-3-benzyloxycarbonylamino-1,3-dideoxy-1-phenylthio-α-D-glucopyranose(43.3 g, 74.7 mmol, 1.2 eq.), which had been dried under the reducedpressure overnight and molecular sieves 4 A (powder, 210 g), which hadbeen dried under the reduced pressure overnight. The mixture was stirredat room temperature under a nitrogen atmosphere for one hr. The reactionmixture was cooled to −15° C. N-Iodosuccinimide (67.2 g, 299 mmol, 4.8eq.) and trifluoromethanesulfonic acid (1.1 mL, 12.5 mmol, 0.2 eq.) wereadded thereto, and the mixture was stirred under light shielding at −10°C. for 50 min. N-Iodosuccinimide (33.6 g, 149 mmol, 2.4 eq.) andtrifluoromethanesulfonic acid (0.55 mL, 6.23 mmol, 0.1 eq.) were addedthereto, and the mixture was stirred at −10° C. for additional 50 min.Triethylamine (3.47 mL, 24.9 mmol, 0.4 eq.) was then added to thereaction mixture to stop the reaction. The insolubles were filtered(washed with 1.4 L of chloroform), and the organic layer was washed oncewith a 10% aqueous sodium thiosulfate solution (2.8 L) and once with a5% aqueous sodium hydrogencarbonate solution (2.8 L) and was dried overanhydrous magnesium sulfate. The residue was purified by chromatographyon silica gel to give the title compound. Developingsolvent=chloroform:acetone=7/1→5/1 (134.11 g, 84.2 mmol, yield 68%).

LCMS: m/z 1593 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.22 (d, 1H, J=3.9 Hz), 7.85 (d, 1H, J=7.6 Hz),7.67-7.77 (m, 1H), 7.13-7.56 (m, 40H), 7.05-7.15 (m, 1H), 6.92-7.03 (m,2H), 6.23-6.38 (m, 1H), 5.82 (d, 1H, J=3.2 Hz), 5.62 (s, 1H), 5.40 (t,1H, J=10, 10 Hz), 5.12-5.35 (m, 10H), 4.87 (ABq, 2H, J=12, 12 Hz), 4.95(ABq, 2H, J=11 Hz), 4.67 (q, 1H, J=10, 10, 10 Hz), 4.60 (ABq, 2H, J=12,12 Hz), 3.92-4.58 (m, 13H), 3.37-3.61 (m, 4H), 2.18-2.45 (m, 2H), 2.00(s, 3H), 1.87 (s, 3H), 1.82-1.96 (m, 2H), 1.45-1.68 (m, 2H).

Production Step 4-52″,2′″-Di-O-benzyl-3″-deoxy-2-hydroxyl-3,2′,6′3″-tetra-N-benzyloxycarbonylarbekacin

The compound (0.6422 g, 0.40 mmol) produced in production step 4-4 wasdissolved in a mixed liquid composed of chloroform (12.8 mL) andmethanol (6.4 mL). 0.5 M sodium methoxide (methanol solution, 0.26 mL,0.13 mmol) was added to the solution, and the mixture was stirred atroom temperature for 2 hr. Acetic acid (7.4 μL, 0.13 mmol) was addedthereto, and the mixture was washed with a saturated aqueous sodiumhydrogencarbonate solution (40 mL). The water layer was again extractedwith chloroform (40 mL), and the combined organic layers were dried overmagnesium sulfate. The solvent was removed by distillation under thereduced pressure, and the residue was purified by column chromatographyon silica gel (chloroform:methanol=30:1) to give the title compound(0.5798 g, 0.38 mmol, yield 95%).

LCMS: m/z 1509 [M+H]⁺

¹H-NMR (Pyridine-d5): δ 8.09 (d, 1H, J=7.8 Hz), 7.60-7.70 (m, 1H), 7.57(d, 1H, J=8.8 Hz), 7.15-7.55 (m, 40H), 6.70-7.10 (m, 2H), 6.65-6.75 (m,1H), 6.04 (s, 1H), 5.47-5.58 (m, 3H), 5.15-5.35 (m, 10H), 4.93 (ABq, 2H,Jgem=11, 11 Hz), 4.79 (ABq, 2H, Jgem=12, 12 Hz), 4.61 (ABq, 2H, Jgem=12,12 Hz), 4.05-4.59 (m, 13H), 3.90-3.98 (m, 2H), 3.38-3.60 (m, 4H),2.19-2.48 (m, 2H), 1.85-1.95 (m, 2H), 1.43-1.62 (m, 2H).

Production Step 4-6 2-Hydroxyarbekacin

The compound (290.0 mg, 0.192 mmol) produced in production step 4-5 wasdissolved in 6 mL of a solution of 1,4-dioxane/water/1 N hydrochloricacid=40/19/1. The solution was adjusted to pH 1.54, a palladium blackpowder (145 mg) was added thereto, and the mixture was vigorouslystirred under a hydrogen atmosphere. The pH value of the reaction systemreached 8.50 2 hr after the start of the vigorous stirring. The reactionsystem was adjusted to pH 1.68 by the addition of 1 N hydrochloric acid(860 μl) and was vigorously stirred under a hydrogen atmosphere for 39hr. The palladium black powder was removed by filtration through acotton, and the catalyst was washed with water. The filtrate and thewash liquid were combined and were concentrated to dryness. The residuewas dissolved in 10 mL of water, and the solution was purified by anAmberlite CG-50 column (equilibrated with 0.005 M NH₄OH) to give thetitle compound (0.1165 g, yield 72%).

LCMS: m/z 569 [M+H]⁺

¹H-NMR (26% ND₃-D₂O): δ 5.13 (d, 1H, J=3.5 Hz), 5.03 (d, 1H, J=4 Hz),4.16 (dd, 1H, J=4, 9.5 Hz), 3.98 (m, 1H), 3.83-3.90 (m, 2H), 3.65-3.77(m, 4H), 3.37 (t, 1H, J=10, 10 Hz), 3.34 (t, 1H, J=10, 10 Hz), 3.32 (dd,1H, J=4, 10 Hz), 3.25 (t, 1H, J=10, 10 Hz), 2.97 (t, 1H, J=10, 10 Hz),2.86 (t, 1H, J=10, 10 Hz), 2.82 (m, 1H), 2.70-2.78 (m, 2H), 2.65 (dd,1H, J=5, 13 Hz), 2.62 (dd, 1H, J=7, 13 Hz), 1.86-1.96 (m, 1H), 1.69-1.80(m, 3H), 1.61 (dq, 1H, J=4, 13, 13, 13 Hz), 1.37 (dq, 1H, J=4, 13, 13,13 Hz).

Example 5 1-N—[(S)-3-Amino-2-hydroxypropionyl]-2-hydroxydibekacin

Production Step 5-11-N—[(S)-2-Acetoxy-3-amino-N-benzyloxycarbonylpropionyl]-2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxyneamine

(S)-2-Acetoxy-3-amino-N-benzyloxycarbonylpropanoic acid (21.2 mg)produced in Reference Example 2 and 43.2 mg of2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxy-2-hydroxyneamineproduced in production step 3-6 of Example 3 were dissolved in 0.6 mL oftetrahydrofuran. 2-Propanol (1.3 mL), 0.1 mL of water and 22.4 mg of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM) were added to the solution, and the mixture was stirred at roomtemperature for one hr. The reaction solution was concentrated under thereduced pressure, 20 mL of chloroform was added to the residue, and themixture was washed once with 15 mL of a saturated aqueous sodiumbicarbonate solution and three times with 15 mL of water and was driedover a Glauber's salt and was concentrated to dryness. The residue waspurified by column chromatography on silica gel (3 g,chloroform→chloroform:methanol=→100:1→100:5→10:1) to give title compound(43.0 mg, yield 75%).

Rf value: 0.57 (chloroform:methanol=10:1)

ESIMS: m/z 1084 [M+Na]⁺

¹H-NMR (pyridine-d5): δ 9.53 (d, 1H, J=6.5 Hz), 8.08 (d, 1H, J=7.5 Hz),7.68 (d, 1H, J=7.0 Hz), 7.49 (d, 1H, J=7.5 Hz), 7.21-7.45 (m, 26H), 5.72(d, 1H, J=5.8 Hz), 5.44 (d, 1H, J=12.3 Hz), 5.39 (d, 1H, J=12.1 Hz),5.22-5.37 (m, 7H), 5.17 (d, 1H, J=12.0 Hz), 5.02 (d, 1H, J=10.5 Hz),4.59 (br, 1H), 4.43 (dd, 1H, J=8.9, 9.3 Hz), 4.29-4.39 (m, 3H),4.19-4.22 (m, 2H), 4.08 (dd, 1H, J=7.9, 9.3 Hz), 4.00-4.06 (m, 2H),3.58-3.62 (m, 1H), 3.50-3.53 (m, 1H), 2.01 (br, 2H), 1.75 (s, 3H), 1.62(br, 2H).

Production Step 5-21-N—[(S)-3-Amino-N-benzyloxycarbonyl-2-hydroxypropionyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin

The compound (225 mg) produced in production step 5-1 was dissolved in7.0 mL of methylene chloride. The compound (246 mg) produced inproduction step 4-3 of Example 4 and 675 mg of a molecular sieves 4 Apowder were added to the solution, and the mixture was stirred at roomtemperature for one hr. The stirred mixture was cooled to −20° C., 238mg of N-iodosuccinimide and 5.5 μL of trifluoromethanesulfonic acid wereadded thereto, and the mixture was stirred under light shieldedconditions at room temperature for 3 hr. Triethylamine (55 μL) was addedthereto under ice cooling. The reaction solution was filtered throughCelite, and the insolubles were washed with 30 mL of chloroform. Thesolution thus obtained was washed once with 20 mL of a saturated aqueoussodium bicarbonate solution and twice with 15 mL of a 10% aqueous sodiumthiosulfate solution, was dried over a Glauber's salt, and wasconcentrated to dryness. The residue was purified by columnchromatography on silica gel (16 g, chloroform:ethylacetate=30:1→20:1→chloroform:methanol=20:1→10:1) to give 320 mg of acrude product containing4″,6″-di-O-acetyl-1-N—[(S)-2-acetoxy-3-amino-N-benzyloxycarbonylpropionyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacinas a contemplated product.

Rf value: 0.56 (chloroform:ethyl acetate=2:3): contemplated product

ESIMS: m/z 1553 [M+Na]⁺: contemplated product

The crude product (320 mg) was dissolved in 6.0 mL of methanol. Sodiumborohydride (7.1 mg) was added to the solution under ice cooling, andthe mixture was stirred at room temperature for 1 hr. Acetone (0.5 mL)was added thereto under ice cooling, and the mixture was stirred at roomtemperature for 15 min. The mixture was then diluted with 30 mL ofchloroform, 20 mL of water was added to the diluted solution, followedby separation. The organic layer was dried over a Glauber's salt and wasconcentrated to dryness to give 272 mg of a crude product(4″,6″-di-O-acetyl-1-N—[(S)-2-acetoxy-3-amino-N-benzyloxycarbonylpropionyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin).

Rf value: 0.56 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1553 [M+Na]⁺

The crude product (272 mg) was dissolved in 5.4 mL of chloroform and 2.7mL of methanol. A 28% solution (9 μL) of sodium methoxide in methanolwas added to the solution under ice cooling, and the mixture was stirredat room temperature for 2 hr. Acetic acid (0.1 mL) was added theretounder ice cooling, the mixture was diluted with 20 mL of chloroform, andthe dilution solution was washed with 10 mL of a saturated aqueoussodium bicarbonate solution, was dried over Glauber's salt, and wasconcentrated to dryness. The residue was purified by columnchromatography on silica gel (20 g,chloroform→chloroform:methanol=99:1→97:3) to give the title compound(141 mg, yield 47%).

Rf value: 0.18 (chloroform:ethyl acetate=2:3)

¹H-NMR (pyridine-d5): δ 8.81 (d, 1H, J=6.6 Hz), 8.40 (d, 1H, J=7.5 Hz),8.31 (d, 1H, J=7.4 Hz), 7.91 (br, 1H), 7.65 (d, 1H, J=7.5 Hz), 7.22-7.51(m, 36H), 5.78 (d, 1H, J=5.8 Hz), 5.73 (d, 1H, J=6.1 Hz), 5.44 (d, 1H,J=12.1 Hz), 5.39 (d, 1H, J=12.1 Hz), 5.18-5.38 (m, 10H), 5.11 (m, 1H),5.07 (d, 1H, J=10.5 Hz), 5.01 (d, 1H, J=11.8 Hz), 4.94 (d, 1H, J=10.5Hz), 4.78 (br, 1H), 4.69-4.75 (m, 4H), 4.45 (br, 1H), 4.42 (dd, 1H,J=8.5, 9.2 Hz), 4.37 (m, 1H), 4.33 (m, 1H), 4.29 (dd, 1H, J=7.7, 8.9Hz), 4.26 (dd, 1H, J=7.9, 9.5 Hz), 4.00-4.05 (m, 2H), 3.82-3.89 (m, 2H),3.58-3.62 (m, 1H), 3.49-3.53 (m, 1H), 1.93-2.00 (m, 2H), 1.58 (br, 2H).

ESIMS: m/z 1427 [M+Na]⁺

Production Step 5-31-N—[(S)-3-Amino-2-hydroxypropionyl]-2-hydroxydibekacin

The compound (141 mg) produced in production step 5-2 was dissolved in 7mL of tetrahydrofuran:water:acetic acid (4:1:1). Thereafter, seven dropsof palladium black/water were added as a catalyst, and the mixture wasvigorously stirred at room temperature under a hydrogen atmosphere for13 hr in which the catalyst was replaced once in the 13-hr period. Thereaction solution was filtered through a cotton, and the mother liquorwas concentrated under the reduced pressure. Water was added to theresidue, and the solution was again concentrated to dryness under thereduced pressure. The residue was dissolved in 5 mL of water. Thesolution was neutralized with 0.1 M aqueous ammonia and was charged intoan Amberlite CG-50 column (equilibrated with 0.005 M aqueous ammonia, 5mL), and the column was washed with 0.005 M aqueous ammonia (5 mL).Elution was carried out with 0.10 M→0.25 M→0.50 M→0.75 M aqueous ammonia(each 10 mL). The corresponding fractions were concentrated to drynessto give the title compound (1.5 carbonate 0.75 hydrate, 47 mg (71%)).

Rf value: 0.18 (chloroform:methanol:28% aqueous ammonia:ethanol=4:6:7:2)

¹H-NMR (DCI-D₂O, pD ˜3): δ 5.86 (d, 1H, J=3.4 Hz), 5.17 (d, 1H, J=3.8Hz), 4.56 (dd, 1H, J=4.4, 7.8 Hz), 4.25 (dt, 1H, 4.0, 12.5), 4.16 (t,1H, J=10.1 Hz), 4.13 (t, 1H, J=10.0 Hz), 4.05 (m, 1H), 3.97 (dd, 1H,J=1.9, 8.9 Hz), 3.94 (dd, 1H, J=1.8, 9.6 Hz), 3.88 (dd, 1H, J=3.1, 8.9Hz), 3.77-3.84 (m, 3H), 3.72 (t, 1H, J=10.1 Hz), 3.60-3.64 (m, 1H), 3.49(t, 1H, J=10.5 Hz), 3.37-3.45 (m, 2H), 3.33 (dd, 1H, J=2.1, 8.0 Hz),3.27 (dd, 1H, J=1.9, 6.5 Hz), 3.16 (dd, 1H, J=6.9, 13.4 Hz), 2.07-2.13(m, 2H), 1.92-1.98 (m, 1H), 1.60-1.66 (m, 1H).

¹³C-NMR (DCI-D₂O, pD ˜3): δ 174.06, 98.72, 95.19, 78.33, 74.95, 74.57,72.41, 68.23, 68.06, 67.55, 66.38, 65.89, 60.10, 55.83, 55.50, 55.19,49.07, 42.92, 42.22, 25.65, 20.84

Calcd. for C₂₁H₄₂N₆O₁₁.1.5H₂CO₃.0.75H₂O: C, 40.87; H, 7.09; N, 12.71.Found. C, 40.99; H, 7.07; N, 12.65.

Example 6 1-N—[(S)-5-Amino-2-hydroxypentanoyl]-2-hydroxydibekacin

Production Step 6-11-N—[(S)-5-Amino-2-benzyloxy-N-benzyloxycarbonylpentanoyl]-2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxyneamine

(S)-5-Amino-2-benzyloxy-N-benzyloxycarbonylpentanoic acid (10 mg)produced in Reference Example 3 and 22 mg of2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxy-2-hydroxyneamineproduced in production step 3-6 of Example 3 were dissolved in 0.33 mLof tetrahydrofuran. 2-Propanol (0.70 mL, water 40 μL) and 9 mg of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM) were added to the solution, and the mixture was stirred at roomtemperature for 2 hr. The reaction solution was concentrated under thereduced pressure. Chloroform (20 mL) was added to the residue, and thesolution was washed once with 10 mL of a saturated aqueous sodiumbicarbonate solution and three times with 10 mL of water, was dried overa Glauber's salt and was concentrated to dryness. The residue waspurified by column chromatography on silica gel (5 g,chloroform→chloroform:methanol=99:1→97:3→10:1) to give the titlecompound (22 mg, yield 72%).

Rf value: 0.60 (chloroform:methanol=10:1)

ESIMS: m/z 1160 [M+Na]³⁰

¹H-NMR (pyridine-d5): δ 8.66 (d, 1H, J=8.0 Hz), 8.43 (d, 1H, J=7.2 Hz),8.11 (d, 1H, J=7.8 Hz), 7.87 (br, 1H), 7.70 (br, 1H), 7.60 (d, 2H, J=7.4Hz), 7.39-7.51 (m, 8H), 7.21-7.31 (m, 20H), 5.79 (d, 1H, J=5.8 Hz), 5.41(d, 1H, J=12.5 Hz), 5.38 (d, 1H, J=9.9 Hz), 5.31-5.35 (m, 2H), 5.28 (br,2H), 5.21 (d, 1H, J=12.5 Hz), 5.03-5.19 (m, 4H), 4.78 (d, 1H, J=11.8Hz), 4.65 (q, 1H, J=9.4 Hz), 4.52 (t, 1H, J=9.5 Hz), 4.49 (d, 1H, J=11.8Hz), 4.37 (br, 2H), 4.19-4.22 (m, 1H), 4.17 (t, 1H, J=7.3 Hz), 4.10 (t,1H, J=7.7 Hz), 4.06 (dd, 1H, J=7.7, 9.6 Hz), 3.60-3.67 (m, 1H),3.52-3.55 (m, 1H), 3.34 (m, 2H), 2.13 (br, 2H), 1.99-2.09 (m, 3H), 1.90(dt, 1H, 5.9, 8.0 Hz), 1.62 (br, 2H).

Production Step 6-21-N—[(S)-5-Amino-2-benzyloxy-N-benzyloxycarbonylpentanoyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin

The compound (350 mg) produced in production step 6-1 was dissolved in8.8 mL of methylene chloride. The compound (364 mg) produced inproduction step 4-3 of Example 4 and 1000 mg of a molecular sieves 4 Apowder were added to the solution, and the mixture was stirred at roomtemperature for one hr. The reaction solution was cooled to −20° C., 350mg of N-iodosuccinimide and 8.1 μL of trifluoromethanesulfonic acid wereadded to the cooled solution, and the mixture was stirred under lightshielded conditions at room temperature for 5 hr. Triethylamine (81 μL)was added thereto under ice cooling, and the reaction mixture wasfiltered through Celite. The insolubles were washed with 20 mL ofchloroform. The solution thus obtained was washed once with 15 mL of asaturated aqueous sodium bicarbonate solution and twice with 15 mL of a10% aqueous sodium thiosulfate solution, was dried over a Glauber's saltand was concentrated to dryness. The residue was purified by columnchromatography on silica gel (25 g, chloroform:ethylacetate=4:1→chloroform:methanol=20:1→10:1) to give a 467 mg of a crudeproduct containing4″,6″-di-O-acetyl-1-N—[(S)-5-amino-2-benzyloxy-N-benzyloxycarbonylpentanoyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacinas a contemplated product.

Rf value: 0.51 (chloroform:ethyl acetate=2:3): contemplated product

ESIMS: m/z 1630 [M+Na]⁺: contemplated product

The crude product (467 mg) was dissolved in 9.3 mL of methanol, 20 mg ofsodium borohydride was added under ice cooling at room temperature for1.5 hr. Acetone (1.0 mL) was added thereto under ice cooling, themixture was stirred at room temperature for 15 min, and the mixture wasdiluted with 50 mL of chloroform. Water (25 mL) was added theretofollowed by separation. The organic layer was dried over a Glauber'ssalt and was then concentrated to dryness to give 381 mg of a crudeproduct(4″,6″-di-O-acetyl-1-N—[(S)-5-amino-2-benzyloxy-N-benzyloxycarbonylpentanoyl]-2,2″-di-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin).

Rf value: 0.51 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1630 [M+Na]⁺

The crude product (381 mg) was dissolved in 7.6 mL of chloroform and 3.8mL of methanol. A 28% solution (13 μL) of sodium methoxide in methanolwas added to the solution under ice cooling, and the reaction wasallowed to react at room temperature for 2 hr. Acetic acid (0.1 mL) wasadded thereto under ice cooling, and the mixture was diluted with 25 mLof chloroform. Thereafter, the diluted solution was washed with 15 mL ofa saturated aqueous sodium bicarbonate solution, was dried over aGlauber's salt and was concentrated to dryness. The residue was purifiedby column chromatography on silica gel (30 g,chloroform→chloroform:methanol=99:1→97:3) to give the title compound 229mg (yield 49%).

Rf value: 0.22 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1545 [M+Na]⁺

¹H-NMR (pyridine-d5): δ 8.69 (br, 1H), 8.38 (d, 1H, J=7.9 Hz), 7.65-7.72(m, 2H), 7.60 (br, 4H), 7.20-7.52 (m, 38H), 5.78 (br, 1H), 5.72 (d, 1H,J=6.1 Hz), 5.20-5.39 (m, 13H), 5.09-5.11 (m, 1H), 5.05 (d, 1H, J=9.0Hz), 5.01 (t, 1H, J=8.0 Hz), 4.89 (d, 1H, J=12.1 Hz), 4.68-4.80 (m, 6H),4.48 (d, 1H, J=12.1 Hz), 4.15-4.34 (m, 5H), 4.06 (br, 2H), 3.93 (br,1H), 3.88 (t, 1H, J=8.1 Hz), 3.52-3.59 (m, 1H), 3.31-3.37 (m, 1H),1.87-2.20 (m, 5H), 1.78 (br, 1H), 1.62 (br, 2H).

Production Step 6-31-N—[(S)-5-Amino-2-hydroxypentanoyl]-2-hydroxydibekacin

The compound (229 mg) produced in production step 6-2 was dissolved in9.2 mL of 1,4-dioxane:water:1 N hydrochloric acid (40:19:1), and sixdrops of palladium black/water were added as a catalyst. The mixture wasvigorously stirred at room temperature under a hydrogen gas atmospherefor 14 hr. In this case, the catalyst was replaced once in the 14-hrperiod. Since the progress of the reaction stopped, the reactionsolution was filtered through a cotton, 6 mL of a saturated aqueoussodium bicarbonate solution was added followed by separation with 9 mLof ethyl acetate. The water layer was again extracted with 9 mL of ethylacetate. The organic layers were combined, were dried over a Glauber'ssalt and were concentrated to dryness. The residue (161 mg) wasdissolved in 8 mL of tetrahydrofuran:water:acetic acid (4:1:1).Palladium black/water (nine drops) was added as a catalyst, and themixture was vigorously stirred under a hydrogen gas atmosphere at roomtemperature for 19 hr. In this case, the catalyst was replaced threetimes in the 19-hr period. The reaction solution was filtered through acotton, and the mother liquor was concentrated under the reducedpressure. Thereafter, water was again added, and the mixture was againconcentrated under the reduced pressure. The residue was dissolved in 5mL of water. The solution was neutralized with 0.1 M aqueous ammonia andwas charged into an Amberlite CG-50 column (equilibrated with 0.005 Maqueous ammonia, 5 mL), and the column was washed with 0.005 M aqueousammonia (5 mL). Elution was carried out with 0.10 M→0.25 M→0.50 M→0.75 Maqueous ammonia (each 10 mL). The corresponding fractions wereconcentrated to dryness to give 48 mg (41%) of the title compound (2.25carbonate 2.75 hydrate).

Rf value: 0.20 (chloroform:methanol:28% aqueous ammonia:ethanol=4:6:7:2)

¹H-NMR (26% ND₃-D₂O): δ 5.05 (d, 1H, J=3.3 Hz), 4.95 (d, 1H, J=3.7 Hz),4.02 (dd, 1H, J=3.3, 8.0 Hz), 3.89 (dt, 1H, 2.0, 8.1), 3.81 (t, 1H,J=10.1 Hz), 3.75-3.79 (m, 1H), 3.61-3.70 (m, 4H), 3.29 (t, 1H, J=10.2Hz), 3.24 (t, 1H, J=10.2 Hz), 3.21 (t, 1H, J=10.4 Hz), 3.17 (t, 1H,J=9.8 Hz), 2.88 (t, 1H, J=10.0 Hz), 2.78 (t, 1H, J=10.0 Hz), 2.75 (dt,1H, J=1.7, 12.0 Hz), 2.51-2.59 (m, 4H), 1.70-1.79 (m, 1H), 1.59-1.66 (m,2H), 1.42-1.57 (m, 4H), 1.25-1.32 (m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 178.16, 102.70, 99.16, 83.44, 78.36, 75.58,72.92, 72.59, 72.26, 72.11, 71.48, 70.07, 61.08, 57.19, 56.63, 56.27,54.97, 50.89, 45.99, 41.06, 32.03, 28.42, 26.94

Calcd. for C₂₃H₄₆N₆O₁₁.2.25H₂CO₃.2.75H₂O: C, 39.30; H, 7.31; N, 10.89.Found. C, 39.08; H, 7.14; N, 11.05.

Example 7 1-N—[(S)-6-Amino-2-hydroxyhexanoyl]-2-hydroxydibekacin

Production Step 7-11-N—[(S)-6-Amino-2-benzyloxy-N-benzyloxycarbonylhexanoyl]-2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxyneamine

(S)-6-Amino-2-benzyloxy-N-benzyloxycarbonylhexanoic acid (160 mg)produced in Reference Example 4 and 344 mg of2-benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3,4′-dideoxy-2-hydroxyneamineproduced in production step 3-6 of Example 3 were dissolved in 5.2 mL oftetrahydrofuran. 2-Propanol (10.3 mL), 0.7 mL of water, and 155 mg of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM) were added to the solution, and the mixture was stirred at roomtemperature for 2 hr. The reaction solution was concentrated under thereduced pressure. Chloroform (50 mL) was added to the residue, and themixture was washed once with 25 mL of a saturated aqueous sodiumbicarbonate solution and three times with 20 mL of water, was dried overa Glauber's salt and was concentrated to dryness. The residue waspurified by column chromatography on silica gel (25 g, hexane:ethylacetate=3:1) to give the title compound (355 mg, yield 72%).

Rf value: 0.62 (chloroform:methanol=10:1)

ESIMS: m/z 1174 [M+Na]⁺

Production Step 7-21-N—[(S)-6-Amino-2-benzyloxy-N-benzyloxycarbonylhexanoyl]-2,2″-O-benzyl-3,2,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin

The compound (350 mg) produced in production step 7-1 was dissolved in8.8 mL of methylene chloride. The compound (352 mg) produced inproduction step 4-3 of Example 4 and 1000 mg of a molecular sieves 4 Apowder were added to the solution, and the mixture was stirred at roomtemperature for one hr. The reaction solution was cooled to −20° C.N-Iodosuccinimide (341 mg) and 7.9 μL of trifluoromethanesulfonic acidwere added to the cooled solution.

The mixture was stirred under light shielded conditions at roomtemperature for 3 hr. Triethylamine (79 μL) was added thereto under icecooling. The reaction mixture was filtered through Celite, and theinsolubles were washed with 20 mL of chloroform. The solution thusobtained was washed once with 15 mL of a saturated aqueous sodiumbicarbonate solution and twice with 15 mL of a 10% aqueous sodiumthiosulfate solution, was dried over a Glauber's salt and wasconcentrated to dryness. The residue was purified by columnchromatography on silica gel (25 mg, chloroform:ethylacetate=4:1→chloroform:methanol=20:1→10:1) to give 433 mg of a crudeproduct containing4″,6″-O-acetyl-1-N—[(S)-6-amino-2-benzyloxy-N-benzyloxycarbonylhexanoyl]-2,2″-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacinas a contemplated product.

Rf value: 0.51 (chloroform:ethyl acetate=2:3): contemplated product

ESIMS: m/z 1643 [M+Na]⁺: contemplated product

The crude product (433 mg) was dissolved in 8.7 mL of methanol. Sodiumborohydride (19 mg) was added to the solution under ice cooling, and themixture was stirred at room temperature for 1.5 hr. Acetone (1.0 mL) wasadded thereto under ice cooling, and the mixture was stirred at roomtemperature for 15 min. Thereafter, the reaction mixture was dilutedwith 50 mL of chloroform. Water (25 mL) was added thereto followed byseparation. The organic layer was dried over a Glauber's salt and wasthen concentrated to dryness to give 357 mg of a crude product(4″,6″-O-acetyl-1-N—[(S)-6-amino-2-benzyloxy-N-benzyloxy-carbonylhexanoyl]-2,2″-O-benzyl-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin).

ESIMS: m/z 1643 [M+Na]⁺

The crude product (357 mg) was dissolved in 7.2 mL of chloroform and 3.6mL of methanol. A 28% sodium methoxide-methanol solution (13 μL) wasadded to the solution under ice cooling, and the mixture was stirred atroom temperature for 2 hr. Acetic acid (0.5 mL) was added thereto underice cooling, and the mixture was diluted with 25 mL of chloroform. Thediluted solution was washed with 15 mL of a saturated aqueous sodiumbicarbonate solution and was dried over a Glauber's salt and wasconcentrated to dryness. The residue was purified by columnchromatography on silica gel (28 g,chloroform→chloroform:methanol=99:1→97:3) to give the title compound(207 mg, yield 44%).

Rf value: 0.22 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1560 [M+Na]³⁰

¹H-NMR (pyridine-d5): δ 8.39 (d, 1H, J=7.9 Hz), 7.76 (d, 1H, J=8.9 Hz),7.63 (br, 2H), 7.21-7.55 (m, 42H), 5.81 (br, 1H), 5.74 (br, 1H), 5.40(br, 1H), 5.33-5.38 (m, 3H), 5.36 (t, 1H, J=6.4 Hz), 5.21-5.27 (m, 3H),5.19 (d, 1H, J=11.9 Hz), 4.79-5.08 (m, 8H), 4.75 (d, 1H, J=11.9 Hz),4.69 (d, 1H, J=11.9 Hz), 4.22-4.51 (m, 8H), 3.95-4.13 (m, 4H), 3.50-3.61(m, 2H), 3.27 (br, 2H), 2.02 (br, 2H), 1.94 (br, 2H), 1.42-1.64 (m, 6H).

Production Step 7-31-N—[(S)-6-Amino-2-hydroxyhexanoyl]-2-hydroxydibekacin

The compound (180 mg) produced in production step 7-2 was dissolved in9.0 mL of tetrahydrofuran:water:acetic acid (4:1:1). Palladiumblack/water (nine drops) was added as a catalyst to the solution, andthe mixture was vigorously stirred under a hydrogen gas atmosphere atroom temperature for 33 hr. In this case, the catalyst was replaced sixtimes in the 33-hr period. The reaction solution was filtered through acotton, and the mother liquor was concentrated under the reducedpressure. Thereafter, water was again added to the residue, and thesolution was again concentrated under the reduced pressure. The residuewas dissolved in 5 mL of water. The solution was neutralized with 0.1 Maqueous ammonia and was charged into an Amberlite CG-50 column(equilibrated with 0.005 M aqueous ammonia, 5 mL), and the column waswashed with 0.005 M aqueous ammonia (5 mL). Elution was carried out with0.10 M→0.25 M→0.50 M→0.75 M aqueous ammonia (each 10 mL), and thecorresponding fractions were concentrated to dryness to give the titlecompound (1.5 carbonate trihydrate, 52 mg, yield 60%).

¹H-NMR (26% ND₃-D₂O): δ 5.16 (d, 1H, J=3.2 Hz), 5.05 (d, 1H, J=3.3 Hz),4.05 (dd, 1H, J=3.5, 8.8 Hz), 4.00 (d, 1H, 9.8), 3.90 (t, 1H, J=9.8 Hz),3.82-3.87 (m, 1H), 3.69-3.79 (m, 4H), 3.40 (t, 1H, J=9.2 Hz), 3.29-3.37(m, 2H), 3.27 (t, 1H, J=9.8 Hz), 2.98 (t, 1H, J=10.0 Hz), 2.88 (t, 1H,J=9.8 Hz), 2.85 (dt, 1H, J=4.0, 12.1 Hz), 2.61-2.70 (m, 4H), 1.71-1.83(m, 3H), 1.59-1.69 (m, 2H), 1.33-1.56 (m, 5H).

¹³C-NMR (26% ND3-D₂O): S 178.24, 102.02, 99.07, 83.37, 78.30, 76.65,75.43, 73.62, 72.90, 72.39, 72.07, 71.41, 70.04, 61.06, 56.65, 56.16,54.96, 50.76, 45.95, 41.06, 34.20, 28.40, 26.98, 23.09

Calcd. for C₂₄H₄₈N₆O₁₁.1.5H₂CO₃.3H₂O: C, 41.18; H, 7.72; N, 11.30.

Found. C, 41.20; H, 7.73; N, 11.43.

Example 8 1-N—[(R)-4-Amino-2-hydroxybutyryl]-2-hydroxydibekacin

Production Step 8-12-O-Benzyl-1-N-[2-(R)—O-benzyl-4-benzyloxycarbonylaminobutyryl]-3,2,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxy-2-hydroxyneamine

2-Benzyloxy-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxy-2-hydroxyneamine(420 mg) produced in production step 3-6 of Example 3 and 216 mg of(R)-2-benzyloxy-4-benzyloxycarbonylaminobutyric acid produced inReference Example 5 were dissolved in 18 mL of a solvent(2-propanol:tetrahydrofuran:water=30:10:3).4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(hereinafter referred to as “DMT-MM”) (218 mg) was added to thesolution, and the mixture was stirred at room temperature. DMT-MM (146mg) was added two hr after the start of stirring, and the mixture wasstirred, and, after 3.5 hr, the reaction mixture was concentrated todryness. Chloroform (50 mL) was added to the residue, and the solutionwas washed once with 25 mL of a saturated aqueous sodium bicarbonatesolution and twice with water. The washed solution was dried overmagnesium sulfate and was concentrated to dryness to give 686 mg of acrude product. The crude product was purified by column chromatographyon silica gel (50 g, chloroform→chloroform:methanol=99:1→97:3→95:5→10:1)to give the title compound (528 mg, 85%).

Rf value: 0.71 (chloroform:methanol=10:1)

ESIMS: m/z 1146 [M+Na]⁺

¹H-NMR (pyridine-d5 at 80° C.): δ 8.04 (d, 1H, J=7 Hz), 7.55 (br. d, 1H,J=6 Hz), 7.12-7.50 (31H), 7.00 (br. s, 1H), 6.82 (br. s, 1H), 5.64 (d,1H, J=3 Hz), 5.29 (s, 2H), 5.10-5.27 (4H), 4.95 (s, 2H), 4.50-4.80 (ABq,2H, Jgem=11.5 Hz), 4.36 (t, 1H, J=8, 8 Hz), 4.28 (m, 1H), 4.20-4.25(3H), 4.16 (t, 1H, J=9, 9 Hz), 4.08 (t, 1H, J=9, 9 Hz), 3.98 (m, 1H),3.91 (t, 1H, J=11, 11 Hz), 3.37-3.60 (m, 4H), 2.12-2.30 (m, 2H),1.85-2.00 (m, 2H), 1.43-1.68 (m, 2H).

Production Step 8-22,2″-Di-O-benzyl-1-N-[2-(R)—O-benzyl-4-benzyloxycarbonylaminobutyryl]-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin

The compound (497 mg) produced in production step 4-3 of Example 4 and482 mg of the compound produced in production step 8-1 were dissolved in20 mL of methylene chloride. A molecular sieves 4 A powder (1.445 g) wasadded to the solution, and the mixture was stirred under an argonatmosphere at room temperature for 2 hr. N-Iodosuccinimide (482 mg) and7.6 μL of trifluoromethanesulfonic acid were added to the stirredsolution at −20° C. with stirring. The reaction mixture was returned toroom temperature and was stirred under light shielded conditions for 2hr. The compound (248 mg) produced in production step 4-3 of Example 4,241 mg of N-iodosuccinimide, and 3.8 μL of trifluoromethanesulfonic acidwere added to the reaction solution three hr after the start ofstirring, and, after 4 hr, 27 μL of triethylamine was added theretounder ice cooling. The mixture was filtered through Celite to remove theinsolubles. The insolubles were washed with 40 mL of chloroform. Thecombined organic layers were washed once with 50 mL of a saturatedaqueous sodium bicarbonate solution, three times with 50 mL of a 10%aqueous sodium thiosulfate solution and twice with 50 mL of water, wasdried over a Glauber's salt and was concentrated to dryness. The residuewas purified by column chromatography on silica gel (130 g,chloroform:acetone=10:1→7:1→3:1) to give 530 mg of a crude productcontaining 4″,6″-d i-O-acetyl-2,2″-di-O-benzyl-1-N-[2-(R)—O-benzyl-4-benzyloxycarbonylaminobutyryl]-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacinas a contemplated product.

Rf value: 0.42 (chloroform:ethyl acetate=2:3): contemplated product

ESIMS: m/z 1615 [M+Na]⁺: contemplated product

The crude product (527 mg) was dissolved in 10.5 mL of methanol. Sodiumborohydride (24.2 mg) was added to the solution with stirring under icecooling, and the mixture was stirred at room temperature for one hr.Acetone (0.38 mL) was added thereto under ice cooling, and the mixturewas stirred at room temperature for 30 min. The reaction mixture wasconcentrated under the reduced pressure. The residue was dissolved in 50mL of chloroform, and the solution was washed three times with 25 mL ofwater, was dried over magnesium sulfate and was concentrated to drynessto give 524 mg of a crude product(4″,6″-di-O-acetyl-2,2″-di-O-benzyl-1-N-[2-(R)—O-benzyl-4-benzyloxycarbonylaminobutyryl]-3,2′,6′,3″-tetra-N-benzyloxycarbonyl-2-hydroxydibekacin).

Rf value: 0.42 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1615 [M+Na]⁺

Chloroform (10.5 mL), 5 mL of methanol, and a 28% sodiummethoxide-methanol (21.2 mg)/methanol (0.24 mL) solution were added tothe crude product, and the mixture was stirred at room temperature.Acetic acid (6.3 μL) was added thereto 2.5 hr after the start ofstirring, and the mixture was washed with 30 mL of a saturated aqueoussodium bicarbonate solution. The water layer was again extracted with 30mL of chloroform. The combined organic layers were dried over magnesiumsulfate and was concentrated to dryness. The residue was purified bycolumn chromatography on silica gel (50 g, chloroform:methanol=30:1) togive the title compound (337.5 mg, yield 50%).

Rf value: 0.56 (chloroform:methanol=10:1)

ESIMS: m/z 1531 [M+Na]⁺

¹H-NMR (pyridine-d5 at 80° C.): δ 8.01 (d, 1H, J=8 Hz), 7.57 (br. s,1H), 7.55 (br. s, 1H), 7.31-7.49 (m, 16H), 7.13-7.31 (m, 24H), 6.98 (br.s, 1H), 6.86 (br. s, 1H), 6.80 (br. s, 1H), 5.68 (d, 1H, J=3 Hz), 5.54(d, 1H, J=2.5 Hz), 5.12-5.32 (m, 10H), 4.90 (s, 2H), 4.71-4.95 (ABq, 2H,Jgem=12 Hz), 4.57-4.70 (ABq, 2H, Jgem=12 Hz), 4.58 (t, 1H, J=10, 10 Hz),4.47 (t, 1H, J=9, 9 Hz), 4.43-4.59 (m, 2H), 4.31 (m, 1H), 4.27 (dd, 1H,J=6, 7 Hz), 4.24-4.37 (m, 2H), 3.87-4.14 (m, 6H), 3.54 (m, 2H), 3.40 (m,2H), 2.30 (m, 1H), 2.24 (m, 1H), 1.84-1.96 (m, 2H), 1.44-1.63 (m, 2H).

Production Step 8-31-N—[(R)-4-Amino-2-hydroxybutyryl]-2-hydroxydibekacin

The compound (310 mg) produced in production step 8-2 was dissolved in21.6 mL of tetrahydrofuran-acetic acid-water (4:1:1). Palladium black(20 drops) suspended in water was added to the solution, and the mixturewas stirred at room temperature for 6.5 hr while blowing hydrogen intothe system. The catalyst was removed by filtration, palladium black (20drops) newly suspended in water was added thereto, and the mixture wasstirred at room temperature for 12 hr while blowing hydrogen into thesystem. The catalyst was removed by filtration, palladium black (tendrops) newly suspended in water was added thereto, and the mixture wasstirred at room temperature for 14.5 hr while blowing hydrogen into thesystem. The catalyst was removed by filtration and was washed withwater. The filtrate and the wash liquid were combined and wereconcentrated to dryness. The residue was dissolved in 30 mL of water.The solution was charged into an Amberlite CG-50 column (equilibratedwith 0.005 M aqueous ammonia, 15 mL), and the column was washed with 30mL of 0.005 M aqueous ammonia. Elution was carried out with 0.1 M (30mL)→0.25 M (30 mL)→0.5 M (30 mL)→0.75 M (30 mL)→1.0 M aqueous ammonia(60 mL), and the corresponding fractions were concentrated to dryness togive the title compound (112 mg, 0.5 carbonate dihydrate, 86%).

Rf value: 0.13 (chloroform:methanol:15 M aqueous ammonia (concentratedaqueous ammonia):ethanol=4:6:7:2)

¹H-NMR (26% ND₃-D₂O): δ 5.16 (d, 1H, J=3.5 Hz), 5.01 (d, 1H, J=4 Hz),4.21 (dd, 1H, J=3.5, 9 Hz), 3.98 (m, 1H), 3.93 (t, 1H, J=10.5, 10.5 Hz),3.87 (m, 1H), 3.75 (t, 1H, J=10, 10 Hz), 3.72-3.80 (m, 2H), 3.69 (t, 1H,J=10, 10 Hz), 3.40 (t, 1H, J=10, 10 Hz), 3.35 (t, 1H, J=10, 10 Hz), 3.32(dd, 1H, J=4, 10.5 Hz), 3.28 (t, 1H, J=10, 10 Hz), 2.96 (t, 1H, J=10, 10Hz), 2.88 (t, 1H, J=10, 10 Hz), 2.84 (dt, 1H, J=4, 4, 13 Hz), 2.76 (m,2H), 2.66 (dd, 1H, J=5, 13.5 Hz), 2.64 (dd, 1H, J=7.5, 13.5 Hz), 1.92(m, 1H), 1.70-1.84 (m, 3H), 1.64 (dq, 1H, J=3.5, 13, 13, 13 Hz), 1.39(m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 178.39, 102.05, 99.61, 83.52, 79.21, 75.49,72.90, 72.38, 71.72, 71.47, 70.78, 70.02, 60.97, 56.33, 56.27, 54.97,50.78, 45.98, 37.96, 37.08, 28.41, 26.94. Calcd. forC₂₂H₄₄N₆O₁₁.0.5H₂CO₃.2H₂O. C, 42.51; H, 7.77; N, 13.22. Found, C, 42.51;H, 7.74; N, 13.26.

Example 9 Production of 4″-epi-2-hydroxyarbekacin

Production Step 9-13″-Azido-3,2′,6′,4′″-tetra-N-benzyloxycarbonyl-2,2″,2′″-tri-O-benzyl-3″-deoxy-4″-epi-2-hydroxyarbekacin

2-O-Benzyl-1-N-[2-(S)—O-benzyl-4-benzyloxycarbonylaminobutyryl]-3,2′,6′-tri-N-benzyloxycarbonyl-3′,4′-dideoxy-2-hydroxyneamine(39 mg) produced in production step 3-6 of Example was dissolved in 1.2mL of methylene chloride. The compound (50.3 mg) produced in productionstep 10-5b of Example 10 and 117 mg of a molecular sieves 4 A powderwere added to the solution, and the mixture was stirred at roomtemperature for 30 min. N-Iodosuccinimide (39 mg) and 0.91 oftrifluoromethanesulfonic acid were added thereto at −20° C. withstirring, and the mixture was stirred under light shielded conditions at−20° C. for 3 hr. Triethylamine (1.9 μL) was added thereto at −20° C.,and the reaction mixture was filtered through Celite. The insolubleswere washed with 10 mL of chloroform. The combined organic layers werewashed once with 3 mL of a saturated aqueous sodium bicarbonate solutionand twice with 3 mL of a 10% aqueous sodium thiosulfate solution, wasdried over a Glauber's salt and was concentrated to dryness. The residuewas purified by column chromatography on silica gel (4 g,chloroform:ethyl acetate=30:1→20:1, chloroform:methanol=20:1→10:1) togive 41 mg of a crude product.

Rf value: 0.65 (chloroform:ethyl acetate=2:3)

The crude product (41 mg) was dissolved in 1.2 mL of a mixed liquidcomposed of methylene chloride and a 0.1% solution of sodium methoxidein methanol (methylene chloride:solution=2:1) under ice cooling, and thesolution was returned to room temperature, and a reaction was allowed toproceed for 2 hr. A 50% aqueous acetic acid solution (0.8 mL) was addedthereto under ice cooling. The mixture was diluted with 10 mL ofchloroform, and the diluted solution was washed with 3 mL of a saturatedaqueous sodium bicarbonate solution, was dried over a Glauber's salt andwas concentrated to dryness. The residue was purified by columnchromatography on silica gel (4 g, chloroformchloroform:methanol=99:1→97:3) to give the title compound (24.1 mg,yield 51%).

Rf value: 0.24 (chloroform:ethyl acetate=2:3)

ESIMS: m/z 1423 [M+Na]₊

¹H-NMR (pyridine-d5): δ 8.79 (d, 1H, J=7 Hz), 8.43 (d, 1H, J=9 Hz), 7.90(t, 1H, J=6, 6 Hz), 7.76 (m, 1H), 7.18-7.71 (m, 35H), 7.04 (br. s, 1H),6.59 (br. s, 1H), 6.07 (d, 1H, J=3.5 Hz), 5.74 (d, 1H, J=3 Hz),5.06-5.44 (ABq, 2H, Jgem=12 Hz), 5.24-5.41 (m, 4H), 5.04 (br. s, 1H),4.70-4.80 (m, 3H), 4.57 (dd, 1H, J=4, 10 Hz), 4.36-4.51 (m, 4H),4.18-4.35 (m, 2H), 4.00-4.15 (m, 2H), 3.68 (m, 1H), 3.60 (m, 2H), 3.51(m, 1H), 2.48 (m, 1H), 2.34 (m, 1H), 1.95 (m, 2H), 1.61 (m, 2H).

Production Step 9-2 4″-Epi-2-hydroxyarbekacin

Liquid ammonia (about 5 mL) was reservoired at −50° C. in an egg-planttype flask containing 24 mg of the compound produced in production step9-1. Metallic sodium (31 mg) was added thereto at −50° C., and themixture was vigorously stirred with a glass stirrer bar for 2 hr. Solidammonium chloride was gradually added until the color of radicalsdisappeared. The system was returned to room temperature to evaporateammonia. Finally, the contents of the flask were concentrated to drynessby an evaporator. Water (2.4 mL) was added to the residue, and thesolution was adjusted to pH 7 by the addition of 1 M aqueous ammonia.The neutralized solution was charged into an Amberlite CG-50 column(equilibrated with 0.005 M aqueous ammonia, 3 mL), and the column waswashed with 0.005 M aqueous ammonia (6 mL). Elution was carried out with0.1 M→0.2 M→0.3 M→0.5 M→0.8 M aqueous ammonia (each 6 mL). Thecorresponding fractions were concentrated to dryness to give 7.3 mg ofthe title compound (yield 53%, 2.5 carbonate 4.5 hydrate).

Rf value: 0.16 (chloroform:methanol:15 M aqueous ammonia (concentratedaqueous ammonia):ethanol=4:6:7:2)

¹H-NMR (26% ND₃-D₂O): δ 5.08 (br. s, 1H), 5.00 (br. s, 1H), 4.13 (m,2H), 3.75-3.90 (m, 3H), 3.50-3.75 (m, 4H), 3.47 (br. d, 1H, J=10 Hz),3.20-3.40 (m, 2H), 2.72-2.86 (m, 3H), 2.68 (m, 2H), 2.58 (br. s, 2H),1.83 (m, 1H), 1.48-1.75 (m, 3H), 1.31 (m, 1H).

Calcd. for C₂₂H₄₄N₆O₁₁.2.5H₂CO₃.4.5H₂O. C, 36.57; H, 7.26; N, 10.44.Found, C, 36.65; H, 7.01; N, 10.57.

Example 10 5-Epi-2-hydroxyarbekacin

Production Step 10-1 Methyl3-azido-6-O-benzoyl-2-O-benzyl-3-deoxy-D-glucopyranoside

Methyl 3-azido-2-O-benzyl-3-deoxy-D-glucopyranoside (93 mg) wasdissolved in 0.84 mL of pyridine. Benzoyl chloride (45 μL) was added tothe solution at −20° C. with stirring, and the mixture was stirred at−20° C. for one hr. Water (14 μL) was added thereto, and the mixture wasstirred at −20° C. for 30 min. The contents were concentrated todryness. Chloroform (10 mL) was added thereto, and the mixture waswashed three times with 5 mL of a saturated aqueous sodium bicarbonatesolution, three times with 5 mL of a 5% aqueous potassium bisulfatesolution and three times with 5 mL of water, was dried over a Glauber'ssalt and was concentrated under the reduced pressure. The residue waspurified by column chromatography on silica gel (25 g, hexane:ethylacetate=3:1) to give the title compound (97 mg, yield 78%).

Rf value: 0.72 (chloroform:methanol=20:1)

α Form

¹H-NMR (CDCl₃): δ 7.30-8.10 (m, 10H), 4.62-4.79 (ABq, 2H, Jgem=12 Hz,),4.73 (dd, 1H, J=4, 12 Hz), 4.60 (d, 1H, J=4 Hz), 4.45 (dd, 1H, J=3, 12Hz), 3.87 (ddd, 1H, J=3, 4, 10 Hz), 3.85 (t, 1H, J=10, 10 Hz), 3.39 (s,3H), 3.38 (dd, 1H, J=4, 10 Hz), 3.31 (dt, 1H, J=4, 10 Hz), 2.95 (d, J=4Hz).

β Form

¹H-NMR (CDCl₃): δ 7.30-8.10 (m, 10H), 4.70-4.91 (ABq, 2H, Jgem=12 Hz),4.74 (dd, 1H, J=4, 12 Hz), 4.53 (dd, 1H, J=4, 12 Hz), 4.38 (d, 1H, J=8Hz), 3.59 (s, 3H), 3.57 (dt, 1H, J=4, 4, 12 Hz), 3.50 (t, 1H, J=10, 10Hz), 3.37 (dt, 1H, J=4, 10 Hz), 3.26 (dd, 1H, J=8, 10 Hz).

Production Step 10-2 Methyl3-azido-6-O-benzoyl-2-O-benzyl-3-deoxy-4-O-trifluoromethanesulfonyl-D-glucopyranoside

The compound (3.30 g) produced in production step 10-1 was dissolved in40 mL of methylene chloride. Pyridine (4.8 mL) was added to thesolution, 3.4 mL of anhydrous trifluoromethanesulfonic acid was addedthereto at −20° C. with stirring, and the mixture was stirred at −20° C.for one hr. Methanol (0.81 mL) was added, and the mixture was stirred at−20° C. for 20 min. Chloroform (300 mL) was added thereto, and themixture was washed three times with 170 mL of an ice cooled saturatedaqueous sodium bicarbonate solution, three times with 170 mL of an icecooled 5% aqueous potassium bisulfate solution and three times with 170mL of ice cooled semisaturated brine. The washed solution was dried overa Glauber's salt under ice cooling and was concentrated to dryness underice cooling to give the title compound (3.60 g, 99%). This compound wasunstable and hence was immediately used in the next reaction.

Rf value: 0.45 (hexane:ethyl acetate=3:1)

Production Step 10-3 Methyl3-azido-4-O-acetyl-6-O-benzoyl-2-O-benzyl-3-deoxy-D-galactopyranoside

The compound (3.60 g) produced in production step 10-2 was dissolved in33 mL of N,N-dimethylformamide. Cesium acetate (7.66 g) was added to thesolution, and the mixture was allowed to react at room temperature forone hr. Ethyl acetate (330 mL) was added thereto, and the mixture waswashed twice with 180 mL of water, twice with 180 mL of a saturatedaqueous sodium bicarbonate solution and twice with semisaturated brine,was dried over a Glauber's salt and was concentrated to dryness to givethe title compound (3.72 g, yield 99%).

Rf value: 0.40 (hexane:ethyl acetate=3:1)

α Form

¹H-NMR (CDCl₃): δ 7.20-8.10 (m, 10H), 5.47 (d, 1H, J=4 Hz), 4.72-4.95(ABq, 2H, Jgem=12 Hz,), 4.25 (dd, 1H, J=7, 11 Hz), 4.69 (d, 1H, J=4 Hz),4.38 (ddd, 1H), 4.21 (dd, 1H, J=3, 7 Hz), 3.82 (dd, 1H, J=4, 11 Hz),4.01 (dd, 1H, J=5, 11 Hz), 3.65 (s, 3H), 2.38 (s, 3H).

β Form

¹H-NMR (CDCl₃): δ 7.20-8.10 (m, 10H), 5.45 (d, 1H, J=4 Hz), 4.62-4.82(ABq, 2H, Jgem=12 Hz), 4.50 (dd, 1H, J=7, 12 Hz), 4.39 (d, 1H, J=8 Hz),4.21 (ddd, 1H), 3.94 (t, 1H, J=7, 7 Hz), 3.64 (dd, 1H, J=8, 10 Hz), 3.61(dd, 1H, J=4, 10 Hz), 3.39 (s, 3H), 2.16 (s, 3H).

Production Step 10-43-Azido-1,4-di-O-acetyl-6-O-benzoyl-2-O-benzyl-3-deoxy-D-galactopyranose

The compound (3.72 g) produced in production step 10-3 was dissolved in74 mL of acetic acid:acetic anhydride:sulfuric acid (25:25:1), and thesolution was stirred at room temperature for 3 hr. Chloroform (740 mL)was added thereto, and the mixture was washed three times with 350 mL ofa saturated aqueous sodium bicarbonate solution and three times with 350mL of water, was dried over a Glauber's salt and was concentrated todryness to give the title compound (3.66 g, yield 93%).

Rf value: 0.32 (hexane:ethyl acetate=3:1)

α Form

¹H-NMR (CDCl₃): δ 7.30-8.10 (m, 10H), 6.46 (d, 1H, J=4 Hz), 5.55 (dd,1H), 4.64-4.73 (ABq, 2H, Jgem=12 Hz), 4.40 (dd, 1H, J=7, 11 Hz), 4.35(m, 1H), 4.19 (dd, 1H, J=6, 11 Hz), 3.96 (m, 2H), 2.16 (s, 3H), 2.14 (s,3H).

β Form

¹H-NMR (CDCl₃): δ 7.30-8.10 (m, 10H), 5.69 (d, J=8 Hz), 5.51 (dd, 1H),4.74-4.86 (ABq, 2H, Jgem=12 Hz), 4.42 (m, 1H, J=7, 11 Hz), 4.25 (dd, 1H,J=7, 11 Hz), 4.11 (m, 1H), 3.79 (dd, 1H, J=8, 10 Hz), 3.72 (dd, 1H, J=3,10 Hz), 2.19 (s, 3H), 2.09 (s, 3H).

Production Step 10-5a3-Azido-4-O-acetyl-6-O-benzoyl-2-O-benzyl-1-bromo-3-deoxy-α-D-galactopyranose

The compound (25 mg) produced in production step 10-4 was dissolved in0.5 mL of a solvent (methylene chloride:ethyl acetate=9:1). Titaniumtetrabromide (29 mg) was added under ice cooling with stirring. Themixture was returned to room temperature and was stirred for one hr. Thereaction solution was ice cooled. Ice cooled methylene chloride (2 mL)was added to the cooled solution, and the mixture was washed six timeswith 2 mL of ice cooled water, was dried over a Glauber's salt and wasconcentrated to dryness to give the title compound (27 mg, yield 83%).This compound was unstable and, hence, was immediately used in the nextreaction.

Rf value: 0.82 (hexane:ethyl acetate=3:2)

¹H-NMR (CDCl₃): δ 7.15-8.10 (m, 10H), 6.49 (d, 1H, J=4 Hz), 5.54 (dd,1H, J=2, 4 Hz), 4.45 (dd, 1H, J=7, 11 Hz), 4.39-4.43 (ABq, 2H, Jgem=7Hz), 4.33 (ddd, 1H), 4.27 (dd, 1H, J=6, 11 Hz), 4.06 (dd, 1H, J=4, 11Hz), 3.74 (dd, 1H, J=4, 11 Hz), 2.16 (s, 3H).

Production Step 10-5b3-Azido-4-O-acetyl-6-O-benzoyl-2-O-benzyl-1,3-dideoxy-1-thiophenyl-α-D-galactopyranose

The compound (300 mg) produced in production step 10-4 was dissolved in5 mL of methylene chloride. Phenylthiotrimethylsilane (353 μL) and 135μL of trimethylsilyl trifluoromethanesulfonate were added to thesolution, and the mixture was refluxed with stirring. The reactionsolution was ice cooled six hr after the start of the reflux. Ice cooledmethylene chloride (24 mL) was added to the cooled solution, and themixture was washed twice with 10 mL of an ice cooled 5% aqueous sodiumhydroxide solution and twice with 10 mL of ice cooled water, was driedover a Glauber's salt and was concentrated to dryness, followed byrecrystallization with ethyl acetate/hexane to give the title compound(204 mg, yield 62%).

Rf value: 0.33 (hexane:ethyl acetate=4:1)

¹H-NMR (CDCl₃): δ 7.0-8.0 (m, 15H), 5.76 (d, 1H, J=5.5 Hz), 5.52 (d, 1H,J=3 Hz), 4.82 (br. t, 1H, J=6.5, 6.5 Hz), 4.69-4.80 (ABq, 2H, J=11 Hz),4.36 (dd, 1H, J=7.5, 11.5 Hz), 4.25 (dd, 1H, J=5, 11.5 Hz), 4.17 (dd,1H, J=5.5, 10.5 Hz), 3.92 (dd, 1H, J=3, 10.5 Hz), 2.16 (s, 3H).

Production Step 10-61,3,2′,6′,3″-Penta-N-tert-butoxycarbonyl-2-hydroxygentamicin C1a

2-Hydroxygentamicin C1a (10.0 g) (2.5 sulfate) was dissolved in 140 mLof water. Triethylamine (30 mL) was added to the solution. A solution(180 mL) of 28.1 g of di-tert-butyl dicarbonate in 1,4-dioxane wasadded, and the mixture was stirred at 60° C. for 2 hr. Concentratedaqueous ammonia (17 mL) was added thereto, and the mixture was stirredat 60° C. for 30 min. The reaction mixture was returned to roomtemperature and was concentrated to dryness. Water (1 L) was added tothe residue, and the solution was stirred overnight. The resultantprecipitate was collected by filtration, was washed with water, and wasdried under the reduced pressure to give the title compound (12.4 g,yield 91%).

Rf value: 0.73 (lower layer part of chloroform:methanol:15 M aqueousammonia (concentrated aqueous ammonia)=1:1:1 was used)

ESIMS: m/z 988 [M+Na]⁺

Production Step 10-72,2″-Di-O-acetyl-1,3,2′,6′,3″-penta-N-tert-butoxycarbonyl-2-hydroxygentamicinC1a

The compound (12.4 g) produced in production step 10-6 was dissolved in250 mL of pyridine. Acetic anhydride (36.3 mL) was added to the solutionunder ice cooling, and the mixture was returned to room temperature andwas allowed to react. After the elapse of 88 hr from the start of thereaction, 31.3 mL of methanol was added to the reaction mixture underice cooling, and the mixture was stirred under ice cooling for 30 min.The reaction mixture was concentrated to dryness. Chloroform (1.2 L) wasadded to the residue, and the solution was washed three times with 600mL of a saturated aqueous sodium bicarbonate solution, three times with600 mL of a 5% aqueous potassium bisulfate solution and once with 600 mLof water, was dried over a Glauber's salt and was concentrated under thereduced pressure to give the title compound (13.8 g, quantitative).

Rf value: 0.81 (chloroform:methanol=10:1)

ESIMS: m/z 1072 [M+Na]⁺

Production Step 10-82,2″-Di-O-acetyl-1,3,2′,6′,3″-penta-N-tert-butoxycarbonyl-4″-eno-5-O-methanesulfonyl-2-hydroxygentamicinC1a

The compound (13.5 g) produced in production step 10-7 was dissolved in270 mL of methylene chloride. 4-Dimethylaminopyridine (23.6 g) and 10.9mL of methanesulfonyl chloride were added to the solution under icecooling, and the mixture was returned to room temperature and wasstirred for 45 hr. The reaction mixture was diluted with 1 L ofchloroform, and the diluted solution was washed three times with 600 mLof a saturated aqueous sodium bicarbonate solution, three times with 600mL of a 5% aqueous potassium bisulfate solution and twice with 600 mL ofwater, was dried over a Glauber's salt and was then concentrated underthe reduced pressure to give 19.3 g of a crude product. The crudeproduct was purified by column chromatography on silica gel (200 g,chloroform→chloroform:methanol=50:1) to give the title compound (10.8 g,yield 76%).

Rf value: 0.44 (chloroform:methanol=30:1)

ESIMS: m/z 1132 [M+Na]₊

Production Step 10-92,5,2″-Tri-O-acetyl-1,3,2′,6′,3″-penta-N-tert-butoxycarbonyl-4″-eno-5-epi-2-hydroxygentamicinC1a

The compound (10.5 g) produced in production step 10-8 was dissolved in105 mL of N,N-dimethylformamide. Cesium acetate (16.8 g, dried at 120°C. for 6 hr) was added to the solution, and the mixture was stirred at100° C. for 16 hr. The reaction mixture was diluted with 1.1 L of ethylacetate, was washed once with 300 mL of water, twice with 300 mL ofsaturated brine, was dried over a Glauber's salt and was concentrated todryness to give the title compound (10.1 g, yield 99%).

Rf value: 0.36 (chloroform:methanol=30:1)

ESIMS: m/z 1096 [M+Na]⁺

Production Step 10-101,3,2,6′,3″-Penta-N-tert-butoxycarbonyl-4″-eno-5-epi-2-hydroxygentamicinC1a

A 1% solution (240 mL) of sodium methoxide in methanol was added to thecompound (10.1 g) produced in production step 10-9, and the mixture wasallowed to react at room temperature for 41 hr. The reaction mixture wasneutralized with Dowex 50 W×2 (H⁺ form, substituted with methanol). Theresin was removed by filtration, and the filtrate was concentrated todryness to give the title compound (8.2 g, yield 92%).

Rf value: 0.24 (chloroform:methanol=30:1)

ESIMS: m/z 970 [M+Na]³⁰

Production Step 10-11 3′,4′-Dideoxy-5-epi-2-hydroxyneamine

6 M hydrochloric acid-methanol (1:1) (164 mL) was added to the compound(8.2 g) produced in production step 10-10, and the mixture was allowedto react at room temperature for 7 hr. The reaction mixture was furtherallowed to react at 70° C. for 14 hr. The reaction solution was adjustedto pH 6.6 by the addition of 4 M sodium hydroxide under ice cooling. Thesolution was then diluted with 1.3 L of water, and the diluted solutionwas charged into an Amberlite CG-50 (equilibrated with 0.005 M aqueousammonia) column (900 mL), and elution was successively carried out with0.005 M→0.1 M→0.2 M→0.3 M→0.4 M→0.5 M aqueous ammonia to give the titlecompound (1.54 g, 41% as dicarbonate).

Rf value: 0.16 (chloroform:methanol:15 M aqueous ammonia (concentratedaqueous ammonia):water=1:4:1:1).

¹H-NMR (26% ND3-D₂O): δ 4.90 (d, 1H, J=3 Hz), 4.16 (s, 1H), 3.76 (m,1H), 3.43 (d, 1H, J=10 Hz), 3.29 (d, 1H, J=10 Hz), 3.04 (t, 1H, J=10, 10Hz), 3.00 (t, 1H, J=10, 10 Hz), 2.90 (t, 1H, J=10, 10 Hz), 2.76 (m, 1H),2.61 (dd, 1H, J=4.5, 13.5 Hz), 2.57 (dd, 1H, J=7, 13.5 Hz), 1.6-1.75 (m,3H), 1.35 (m, 1H).

¹³C-NMR (26% ND3-D₂O): δ 96.51, 75.99, 75.31, 72.47, 71.09, 68.40,54.32, 53.38, 50.32, 45.84, 28.35, 27.01.

Production Step 10-123′,4′-Dideoxy-5-epi-1,3,2′,6′-tetra-N-tosyl-2-hydroxyneamine

The compound (202 mg, 0.470 mmol, calculated as dicarbonate) produced inproduction step 10-11 was dissolved in 2.0 mL of water. Sodium carbonate(421 mg) was added to the solution under ice cooling. 1,4-Dioxane (4.0mL) and 541 mg of tosyl chloride were added thereto, and the mixture wasreturned to room temperature and was allowed to react. After the elapseof 2 hr from the start of the reaction, 20 mL of water was added to thereaction mixture. The mixture was extracted three times with 10 mL ofchloroform, was dried over a Glauber's salt and was concentrated todryness. The residue was purified by column chromatography on silica gel(10 g, chloroform:methanol=29:1) to give the title compound (383 mg,88%).

Rf value: 0.43 (chloroform:methanol=10:1)

ESIMS: m/z 945 [M+Na]⁺

¹H-NMR (pyridine-d5): δ 9.41 (d, 1H, J=7 Hz), 8.88 (d, 1H, J=9 Hz), 8.57(t, 1H, J=6, 6 Hz), 8.31 (br., 1H), 7.89-8.15 (m, 8H), 6.96-7.25 (m,8H), 5.22 (d, 1H, J=3 Hz), 4.86 (m, 1H), 4.73 (q, 1H, J=10, 10, 10 Hz),4.60 (br. s, 1H), 4.43 (dt, 1H, J=7, 10, 10 Hz), 4.00 (dd, 1H, J=2, 10.5Hz), 3.86 (t, 1H, J=10, 10 Hz), 3.77 (dd, 1H, J=2, 10.5 Hz), 3.72 (m,1H), 3.15-3.32 (m, 2H), 2.26 (m, 1H), 2.09, 2.13, 2.19, 2.21 (each s,each 3H), 1.51-1.69 (m, 3H).

Production Step 10-134″,6″-Di-O-acetyl-3″-azido-2″-O-benzyl-3″-deoxy-5-epi-1,3,2′,6′-tetra-N-tosyl-2-hydroxydibekacin

Process A: A solution of the compound (372 mg) produced in productionstep 1-5a of Example 1 in 21 mL of methylene chloride was added to thecompound (711 mg) produced in production step 10-12. Drierite (2.16 g)was added thereto, and the mixture was stirred at room temperature for 3hr. Mercuric cyanide (795 mg) was added thereto, and the mixture wasstirred under light shielded conditions at room temperature for 104 hr.The reaction mixture was filtered through Celite to remove theinsolubles. The insolubles were washed with 50 mL of chloroform. Thecombined organic layers were washed twice with 35 mL of a saturatedaqueous sodium bicarbonate solution, twice with 35 mL of a 10% aqueoussodium iodide solution and once with 35 mL of water, were dried over aGlauber's salt and were then concentrated to dryness. The residue waspurified by column chromatography on silica gel (10 g, chloroformchloroform:ethyl acetate=9:1→1:1→2:3) to give the title compound (426mg, yield 44%). In this case, 385 mg (54%) of the starting compound wasrecovered.

Process B: The compound (20.5 mg) produced in production step 10-12 and11.4 mg of the compound produced in production step 1-5b of Example 1were dissolved in 0.4 mL of methylene chloride. A molecular sieves 4 Apowder (63 mg) was added to the solution, and the mixture was stirred atroom temperature for one hr. N-Iodosuccinimide (5.9 mg) and a solutionof 0.6 μL of trifluoromethanesulfonic acid in 0.1 mL of methylenechloride were added thereto at −20° C. with stirring, followed bystirring at −20° C. under light shielded conditions for 15 hr. Thereaction mixture was filtered through Celite to remove the insolubles.The insolubles were washed with 2 mL of chloroform. The combined organiclayers were washed twice with 2 mL of a saturated aqueous sodiumbicarbonate solution, twice with 2 mL of a 10% aqueous sodiumthiosulfate solution and twice with 2 mL of water, were dried over aGlauber's salt and were then concentrated to dryness. The residue waspurified by column chromatography on silica gel (10 g,chloroform→chloroform:ethyl acetate=19:1→9:1→1:1→2:3) to give the titlecompound (7.7 mg, yield 27%). In this case, 7.9 mg (38%) of the startingcompound was recovered.

Rf value: 0.19 (chloroform:ethyl acetate=5:2)

ESIMS: m/z 1306 [M+Na]⁺

¹H-NMR (pyridine-d5): δ 8.99 (d, 1H, J=9 Hz), 8.72 (m, 1H), 8.57 (t, 1H,J=6, 6 Hz), 8.53 (m, 1H), 7.05-8.05 (m, 21H), 5.70 (d, 1H, J=3.5 Hz),5.49 (d, 1H, J=3 Hz), 5.29 (t, 1H, J=10, 10 Hz), 4.84-5.20 (ABq, 2H,Jgem=12 Hz), 5.13 (br. s, 1H), 4.86 (m, 1H), 4.73 (m, 1H), 4.65-4.77 (m,3H), 4.55 (dd, 1H, J=4, 13.5 Hz), 4.12-4.21 (m, 2H), 4.16 (t, 1H, J=10,10 Hz), 3.92 (t, 1H, J=10, 10 Hz), 3.79 (dd, 1H, J=3.5, 10 Hz), 3.63 (m,1H), 3.27 (m, 2H), 2.20 (m, 1H), 2.14, 2.17, 2.21, 2.23 (each s, each3H), 2.00, 2.05 (each s, each 3H), 1.49-1.57 (m, 3H).

Production Step 10-143″-Azido-2″-O-benzyl-3″-deoxy-5-epi-1,3,2′,6′-tetra-N-tosyl-2-hydroxydibekacin

A 0.5% solution (13.4 mL) of sodium methoxide in methanol was added tothe compound (673 mg) produced in production step 10-13, and the mixturewas allowed to react at room temperature for one hr. The reactionmixture was neutralized with Dowex 50 W×2 (H⁺ form, substituted withmethanol). The resin was removed by filtration, and the filtrate wasconcentrated to dryness to give the title compound (593 mg, 94%).

Rf value: 0.21 (chloroform:ethyl acetate=1:1)

ESIMS: m/z 1222 [M+Na]³⁰

Production Step 10-15 5-Epi-hydroxydibekacin

Liquid ammonia (120 mL) was reservoired at −50° C. in an egg-plant typeflask containing 593 mg of the compound produced in production step10-14. Metallic sodium (980 mg) was added at −50° C., and the mixturewas vigorously stirred with a glass stirrer bar for 2 hr. Methanol wasgradually added until the color of radicals disappeared. The mixture wasreturned to room temperature to evaporate ammonia and was finallyconcentrated to dryness by an evaporator. Water (43 mL) was added to theresidue, and the solution was adjusted to pH 4 to 5 with Dowex 50 W×2(H⁺ form). The contents of the flask including the resin as such wereadded to a column packed with 15 mL of the same resin. The column waswashed with 160 mL of water, and elution was carried out with 1 Maqueous ammonia (cut 80 mL). A ninhydrin-positive fraction (Fr 2) wasconcentrated to dryness to give 299 mg of a crude product. The crudeproduct was brought to an aqueous solution (60 mL). The aqueous solutionwas charged into a CM-Sephadex C-25 column (equilibrated with 0.005 Maqueous ammonia, 60 mL), and the column was washed with water (120 mL).Elution was carried out with 0.05 M (300 mL)→0.2 M aqueous ammonia (675mL, cut 12 mL). The corresponding fraction (Fr 38-50) was concentratedto dryness to give the title compound (183 mg, yield 67.5%, asmonocarbonate.monohydrate).

Rf value: 0.29 (1-butanol:ethanol:chloroform:17% aqueousammonia=4:7:2:7)

¹H-NMR (26% ND₃-D₂O): δ 4.99 (d, 1H, J=4 Hz), 4.92 (d, 1H, J=3 Hz), 4.47(br. s, 1H), 3.84 (br. d, 1H, J=12 Hz), 3.83 (m, 1H), 3.79 (m, 1H), 3.62(dd, 1H, J=7.5, 12.5 Hz), 3.49 (dd, 1H, J=2, 10 Hz), 3.42 (dd, 1H, J=4,10.5 Hz), 3.37 (dd, 1H, J=2, 10 Hz), 3.17 (t, 1H, J=10, 10 Hz), 3.15 (t,1H, J=11, 11 Hz), 3.12 (t, 1H, J=10, 10 Hz), 3.10 (t, 1H, J=10, 10 Hz),3.06 (t, 1H, J=10, 10 Hz), 2.79 (m, 1H), 2.65 (dd, 1H, J=4.5, 13.5 Hz),2.60 (dd, 1H, J=7.5, 13.5 Hz), 1.64-1.78 (m, 3H), 1.37 (m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 102.43, 96.75, 83.11, 76.41, 75.21, 74.00,72.95, 71.40, 71.06, 68.07, 62.13, 55.39, 54.37, 53.69, 50.84, 46.49,28.92, 27.62.

Production Step 10-162′,6′-Di-N-benzyloxycarbonyl-5-epi-2-hydroxydibekacin

Nickel acetate tetrahydrate (478 mg) was added to 224 mg (calculated asmonocarbonate monohydrate) of the compound produced in production step10-15. Methanol (9.0 mL) was added thereto, and the mixture was broughtto a homogeneous solution with an ultrasonic cleaner (2 to 3 min, greencolor). N-Benzyloxycarbonyloxysuccinimide (263 mg) was added by portionsunder ice cooling over a period of 2 min. The mixture was stirred underice cooling for one hr. The mixture was returned to room temperature andwas further stirred for 2.5 hr. The reaction mixture was concentrated todryness. A sodium chloride saturated concentrated aqueous ammonia (15mL) was added to the residue, and the mixture was extracted five timeswith 10 mL of 1-butanol. The extracted butanol layer was concentrated todryness. N,N-Dimethylformamide was added to 1594 mg of the residue, andthe mixture was filtered through Celite. The substance on the Celite waswashed with N,N-dimethylformamide (4 mL×6). The filtrate and the washliquid were concentrated to dryness to give 512 mg of a crude product.

Rf value: 0.41 (lower layer part of chloroform:methanol:15 M aqueousammonia (concentrated aqueous ammonia)=1:1:1 was used)

Production Step 10-172′,6′-Di-N-benzyloxycarbonyl-5-epi-2-hydroxy-3″-N-trifluoroacetyldibekacin

The crude product (512 mg) produced in production step 10-16 wasdissolved in 7.1 mL of anhydrous N,N-dimethylformamide. Ethyltrifluoroacetate (74 μL) was added to the solution under ice coolingwith stirring. The mixture was returned to room temperature and wasstirred for 16.5 hr. The reaction solution was concentrated to drynessto give 609 mg of a product.

Rf value: 0.49 (lower layer part of chloroform:methanol:15 M aqueousammonia (concentrated aqueous ammonia)=1:1:1 was used)

Production Step 10-181-N-(4-Benzyloxycarbonylamino-2-(S)-hydroxybutyryl)-2′,6′-di-N-benzyloxycarbonyl-5-epi-2-hydroxy-3″-N-trifluoroacetyldibekacin

The crude product (609 mg) produced in production step 10-17 wasdissolved in 12 mL of anhydrous tetrahydrofuran. Next, a solution (6 mL)of 219 mg of N-benzyloxycarbonyl-4-amino-2-(S)-hydroxybutyric acidsuccinimide ester in tetrahydrofuran synthesized according to the reportof Kawaguchi et al. (Journal of Antibiotics, Vol. 25, pp. 695-708(1972)) was added dropwise to the solution over a period of 3 min underice cooling with stirring. The mixture was returned to room temperatureand was stirred. After the elapse of 3.5 hr from the start of stirring,a solution (0.92 mL) of 34 mg ofN-benzyloxycarbonyl-4-amino-2-(S)-hydroxybutyric acid succinimide esterin tetrahydrofuran was added to the reaction solution under ice coolingwith stirring, and the mixture was returned to room temperature and wasstirred. After the elapse of 18.5 hr from the start of stirring, thereaction solution was concentrated to dryness. Ethyl acetate (150 mL)was added to the residue, and the mixture was washed twice with 30 mL ofa saturated aqueous sodium bicarbonate solution and twice with 30 mL ofwater and was concentrated to dryness to give 604 mg of the reactionmixture.

Rf value: 0.67 (lower layer part of chloroform:methanol:15 M aqueousammonia (concentrated aqueous ammonia)=1:1:1 was used).

Production Step 10-19 5-Epi-2-hydroxyarbekacin

Tetrahydrofuran (20.5 mL) and 15.4 mL of 3.5 M aqueous ammonia wereadded to the reaction mixture (604 mg) produced in production step10-18, and the mixture was stirred at room temperature for 44 hr. Thereaction solution was concentrated to dryness. Tetrahydrofuran-aceticacid-water (4:1:1) (33 mL) was added to 656 mg of the residue. Further,10 drops of a suspension of palladium black in water were added thereto,and the mixture was stirred at the atmospheric pressure for 5 hr whileblowing hydrogen into the system. Next, palladium black was removed fromthe reaction solution by filtration. The palladium black was washed withwater, and the filtrate and the wash liquid were combined and wereconcentrated to dryness. 2 M aqueous ammonia was added to the residue,and the mixture was allowed to stand at room temperature overnight. Theinsolubles were removed by filtration through a cotton stopper and wereconcentrated to dryness to give 466 mg of a crude product. This crudeproduct was dissolved in 60 mL of water to give an aqueous solution. Theaqueous solution was added to a CM-Sephadex C-25 column (equilibratedwith 0.005 M aqueous ammonia, 60 mL). The column was washed with 120 mLof 0.005 M aqueous ammonia. Elution was carried out with aqueous ammoniawith the concentration being varied from 0.05 M (300 mL) to 0.5 M (600mL), and 0.75 M (600 mL) aqueous ammonia. The corresponding fractionswere concentrated to give the title compound: 5-epi-2-hydroxyarbekacin(65 mg, 2.5 carbonate trihydrate, 20% in four steps).

Rf value: 0.11 (1-butanol:ethanol:chloroform:17% aqueousammonia=4:7:2:7)

¹H-NMR (26% ND₃-D₂O): δ 4.95 (d, 1H, J=4 Hz), 4.92 (d, 1H, J=3 Hz), 4.44(t, 1H, J=2, 2 Hz), 4.17 (t, 1H, J=10, 10 Hz), 4.16 (dd, 1H, J=4, 10Hz), 3.85 (dd, 1H, J=2, 12 Hz), 3.79 (dd, 1H, J=2, 10.5 Hz), 3.78 (m,1H), 3.77 (m, 1H), 3.60 (dd, 1H, J=7, 12 Hz), 3.51 (dd, 1H, J=2, 10.5Hz), 3.34 (t, 1H, J=10, 10 Hz), 3.12 (dd, 1H, J=4, 10 Hz), 3.21 (t, 1H,J=10, 10 Hz), 3.14 (t, 1H, J=10, 10 Hz), 3.02 (t, 1H, J=10, 10 Hz), 2.80(m, 1H), 2.70-2.80 (m, 2H), 2.66 (dd, 1H, J=5, 13.5 Hz), 2.61 (dd, 1H,J=7.5, 13.5 Hz), 1.91 (m, 1H), 1.64-1.80 (m, 4H), 1.37 (m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 178.69, 101.16, 96.93, 78.02, 76.08, 74.06,73.28, 72.82, 71.34, 70.92, 68.35, 62.01, 55.40, 54.18, 53.77, 50.69,46.29, 38.64, 37.58, 28.74, 27.43.

Example 11 5,4″-Diepi-2-hydroxyarbekacin

Production Step 11-13,2,6′,3″,4′″-Penta-N-tert-butoxycarbonyl-5-epi-2-hydroxyarbekacin

The compound (65 mg) produced in production step 10-19 of Example 10 wasdissolved in 0.91 mL of water. Triethylamine (0.16 mL) was added to thesolution, a solution of 175 mg of di-tert-butyl dicarbonate in1,4-dioxane (1.17 mL) was added thereto, and the mixture was stirred at60° C. for 1.5 hr. Concentrated aqueous ammonia (0.11 mL) was addedthereto, and the mixture was stirred at 60° C. for 30 min. The reactionmixture was returned to room temperature and was concentrated todryness. The residue was subjected to azeotropic distillation twice withmethanol to give the title compound (103 mg, quantitative).

Rf value: 0.53 (chloroform:methanol: 15 M aqueous ammonia (concentratedaqueous ammonia)=5:1:0.1)

ESIMS: m/z 1091 [M+Na]³⁰

Production Step 11-23,2′,6′,3″,4″-Penta-N-tert-butoxycarbonyl-4″,6″-O-cyclohexylidene-5-epi-2-hydroxyarbekacin

The compound (103 mg) produced in production step 11-1 was dissolved in2.0 mL of N,N-dimethylformamide. Cyclohexanone di-1-propyl acetal (58μL) and 12.4 mg of p-toluenesulfonic acid were added to the solution,and the mixture was allowed to react at room temperature for one hr. Asaturated aqueous sodium bicarbonate solution (20 mL) was added thereto,and the resultant precipitate was collected by filtration, was washedwith water, and was dried under the reduced pressure to give the titlecompound (103 mg, 88%).

Rf value: 0.53 (chloroform:methanol=10:1)

ESIMS: m/z 1171 [M+Na]⁺

Production Step 11-32,2″,2′″-Tri-O-acetyl-3,2′,6′,3″,4′″-penta-N-tert-butoxycarbonyl-4″,6″-O-cyclohexylidene-5-epi-2-hydroxyarbekacin

The compound (103 mg) produced in production step 11-2 was dissolved in2.1 mL of pyridine. Acetic anhydride (0.13 mL) was added to the solutionunder ice cooling, and the mixture was returned to room temperature andwas allowed to react. After the elapse of 18.5 hr from the start of thereaction, 0.11 mL of methanol was added thereto under ice cooling, andthe mixture was allowed to stand at room temperature for 30 min and wasconcentrated to dryness. Chloroform (10 mL) was added to the residue.The solution was washed twice with 3 mL of a saturated aqueous sodiumbicarbonate solution, twice with 3 mL of a 5% aqueous potassiumbisulfate solution and twice with 3 mL of water and was dried over aGlauber's salt and was concentrated to dryness to give the titlecompound (117 mg, quantitative).

Rf value: 0.39 (chloroform:methanol=15:1)

ESIMS: m/z 1297 [M+Na]³⁰

Production Step 11-42,2″,2″-Tri-O-acetyl-3,2′,6′,3″,4′″-penta-N-tert-butoxycarbonyl-5-epi-2-hydroxyarbekacin

The compound (117 mg) produced in production step 11-3 was dissolved in5.17 mL of chloroform methanol (10:1). A 90% aqueous trifluoroaceticacid solution (0.47 mL) was added to the solution under ice cooling withstirring, and the mixture was returned to room temperature and wasstirred. After the elapse of 30 min from the start of stirring, 5.3 mLof chloroform was added. The mixture was washed once with 3 mL of water,twice with 3 mL of a saturated aqueous sodium bicarbonate solution andtwice with 3 mL of semisaturated brine and was dried over a Glauber'ssalt and was concentrated to dryness to give the title compound (107 mg,quantitative).

Rf value: 0.46 (chloroform:methanol=10:1)

ESIMS: m/z 1217 [M+Na]⁺

Production Step 11-5

2,2″,2′″-Tri-O-acetyl-3,2′,6′,3″,4′″-penta-N-tert-butoxycarbonyl-5-epi-2-hydroxy-6″-O-tritylarbekacin

The compound (83 mg) produced in production step 11-4 was dissolved in1.7 mL of pyridine. 4-Dimethylaminopyridine (25 mg) and 116 mg of tritylchloride were added to the solution, and the mixture was allowed toreact at 65° C. After the elapse of 17 hr from the reaction, the mixturewas returned to room temperature. Methanol (0.08 mL) was added thereto,and the mixture was allowed to stand for one hr. The mixture was thenwas concentrated to dryness. Chloroform (8 mL) was added to the residue.The solution was washed twice with 3 mL of a saturated aqueous sodiumbicarbonate solution, three times with 3 mL of a 5% aqueous potassiumbisulfate solution and three times with 3 mL of water, was dried over aGlauber's salt and was concentrated to dryness. The residue was purifiedby column chromatography on silica gel (3.4 g, toluene:ethylacetate:acetone=6:1:1) to give the title compound (65 mg, 65%).

Rf value: 0.57 (chloroform:methanol=10:1)

ESIMS: m/z 1459 [M+Na]³⁰

Production Step 11-62,2″,4″,2′″-Tetra-O-acetyl-3,2′,6′,3″,4′″-penta-N-tert-butoxycarbonyl-5,4″-diepi-2-hydroxy-6″-O-tritylarbekacin

The compound (112 mg) produced in production step 11-5 was dissolved in2.3 mL of methylene chloride under a nitrogen atmosphere. Pyridine (0.13mL) was added to the solution. Trifluoromethanesulfonic acid anhydride(66 μL) was added thereto under a nitrogen atmosphere at −78° C. withstirring. The mixture was stirred at −20° C. under a nitrogen atmospherefor one hr. Methanol (79 μL) was added thereto at −20° C., and 14 mL ofice cooled chloroform was immediately added thereto. The mixture waswashed twice with 7 mL of an ice cooled 10% aqueous potassium bisulfatesolution, twice with 7 mL of an ice cooled saturated aqueous sodiumbicarbonate solution and twice with 7 mL of ice cooled water, followedby drying over a Glauber's salt under ice cooling for 10 min. The driedsolution was concentrated under the reduced pressure under ice cooling.When the resolution became a syrup, the concentration was stopped.

The resultant syrup was dissolved in 1.1 mL of N,N-dimethylformamide.Cesium acetate (150 mg) (dried at 120° C. for 6 hr) was added to thesolution, and the mixture was stirred under a nitrogen atmosphere atroom temperature for 18 hr. The reaction solution was diluted with 46 mLof ethyl acetate, and the diluted solution was washed once with 11 mL ofwater and three times with 11 mL of semisaturated brine, was dried overa Glauber's salt and was concentrated to dryness. The residue waspurified by column chromatography on silica gel (12 g, toluene:ethylacetate:acetone=4:1:1) to give the title compound (75 mg, yield 65%).

Rf value: 0.35 (toluene:ethyl acetate:acetone=3:1:1)

ESIMS: m/z 1501 [M+Na]³⁰

Production Step 11-73,2′,6′,3″,4′″-Penta-N-tert-butoxycarbonyl-5,4″-diepi-2-hydroxy-6″-O-tritylarbekacin

The compound (75 mg) produced in production step 11-6 was dissolved in2.3 mL of a 0.5% solution of sodium methoxide in methanol, and themixture was allowed to react at room temperature for one hr. Thereaction mixture was neutralized with Dowex 50 W×2 (H⁴ form, substitutedwith methanol), and the resin was removed by filtration. The filtratewas concentrated to dryness to give the title compound (64 mg, 96%).

Rf value: 0.60 (toluene:ethyl acetate:acetone=1:1:1)

Production Step 11-8 5,4″-Diepi-2-hydroxyarbekacin

The compound (64 mg) produced in production step 11-7

was dissolved in 1.6 mL of a 90% aqueous trifluoroacetic acid solutionunder ice cooling. The solution was allowed to react under ice coolingfor 2 hr. The reaction mixture was concentrated to dryness. Water (10mL) was added to the residue, and the solution was washed three timeswith 3 mL of diethyl ether. The water layer was concentrated to dryness.0.005 M aqueous ammonia (10 mL) was added to the residue (pH 6 to 7),and the mixture was added to a CM-Sephadex C-25 column (equilibratedwith 0.005 M aqueous ammonia, 10 mL). The column was washed with 30 mLof 0.005 M aqueous ammonia, and elution was carried out with aqueousammonia with the concentration being varied from 0.2 M (50 mL) to 0.5 M(200 mL). The corresponding fractions were concentrated to give 28.6 mgof the title compound: 5-epi-2-hydroxyarbekacin (73% as 2.5 carbonatetrihydrate).

Rf value: 0.09 (chloroform:methanol: 15 M aqueous ammonia (concentratedaqueous ammonia): ethanol=4:6:7:2)

¹H-NMR (DCI-D₂O, pD˜3): δ 5.44 (1H, J=3.5 Hz), 5.16 (d, 1H, J=4 Hz),4.77 (d, 1H, J=3 Hz), 4.35 (dd, 1H, J=4.5, 9 Hz), 4.31 (t, 1H, J=11, 11Hz), 4.17 (d, 1H, J=3 Hz), 4.14 (m, 1H), 4.13 (d, 1H, J=3, 11 Hz), 4.11(m, 1H), 4.02 (dd, 1H, J=3, 11 Hz), 3.98 (dd, J=4, 11 Hz), 3.81 (t, 1H,J=11, 11 Hz), 3.75-3.80 (m, 2H), 3.71 (t, 1H, J=11, 11 Hz), 3.67 (dd,1H, J=3, 11 Hz), 3.63 (m, 1H), 3.28 (dd, 1H, J=3.5, 13.5 Hz), 3.21 (t,2H, J=7, 7 Hz), 3.11 (dd, 1H, J=7.5, 13.5 Hz), 2.23 (m, 1H), 2.05-2.13(m, 2H), 2.00 (m, 1H), 1.94 (m, 1H), 1.64 (m, 1H).

¹³C-NMR (DCI-D₂O, pD˜3): δ 177.13, 100.42, 90.53, 76.63, 71.90, 70.56,70.02, 68.33, 66.26, 66.20, 66.18, 65.57, 61.72, 53.73, 53.49, 52.59,48.59, 42.96, 37.36, 31.34, 25.86, 21.32.

Calcd. for C₂₂H₄₄N₆O₁₁.2.5H₂CO₃.3H₂O. C, 37.84; H, 7.13; N, 10.81.

Found, C, 37.51; H, 7.49; N, 10.96.

Example 12 5,4″-Diepi-2-hydroxydibekacin

Production Step 12-14″-O-Acetyl-3″-azido-6″-O-benzoyl-2″-O-benzyl-3″-deoxy-5,4″-diepi-1,3,2′,6′-tetra-N-tosyl-2-hydroxydibekacin

A solution of 667 mg of the compound produced in production step 10-5ain 34 mL of methylene chloride was added to the compound (1.16 g)produced in production step 10-12. Drierite (3.43 g) was added thereto,and the mixture was stirred at room temperature for 3 hr. Mercuriccyanide (1.27 g) was added to the reaction solution, and the mixture wasstirred under light shielded conditions at room temperature for 42 hr.The reaction solution was filtered through Celite to remove theinsolubles. The insolubles were washed with 90 mL of chloroform. Thecombined organic layers were washed twice with 60 mL of a saturatedaqueous sodium bicarbonate solution, twice with 60 mL of a 10% aqueoussodium iodide solution and once with 60 mL of water, were dried over aGlauber's salt and were concentrated to dryness. The residue waspurified by column chromatography on silica gel (40 g,chloroform→chloroform:ethyl acetate=1:1→ethyl acetate) to give the titlecompound (529 mg, 31%). In this case, 423 mg (36%) of the startingcompound was recovered.

Rf value: 0.20 (chloroform:ethyl acetate=5:2)

ESIMS: m/z 1368 [M+Na]³⁰

¹H-NMR (pyridine-d5): δ 9.02 (d, 1H, J=7 Hz), 8.83 (d, 1H, J=7 Hz), 8.50(m, 1H), 8.48 (t, 1H, J=6, 6 Hz), 7.03-8.28 (m, 26H), 6.00 (d, 1H, J=3Hz), 5.85 (d, 1H, J=2 Hz), 5.58 (br. s, 1H), 4.81-5.26 (ABq, 2H, Jgem=12Hz), 5.20 (br. s, 1H), 5.07 (m, 1H), 4.80-4.95 (m, 4H), 4.77 (t, 1H,J=10, 10 Hz), 4.72 (m, 1H), 4.68 (t, 1H, J=10, 10 Hz), 4.35 (m, 1H),4.26 (br. d, 1H, J=11 Hz), 4.23 (dd, 1H, J=4, 10 Hz), 3.90 (t, J=10, 10Hz), 3.63 (m, 1H), 3.29 (m, 2H), 2.26 (m, 1H), 2.00, 2.07, 2.15, 2.18,2.22 (each s, each 3H), 1.48-1.64 (m, 3H).

Production Step 12-2 5,4″-Diepi-2-hydroxydibekacin

A 0.5 M solution (12 mL) of sodium methoxide in methanol was added tothe compound (529 mg) produced in production step 12-1, and the mixturewas allowed to react at room temperature for 2 hr. The reaction mixturewas neutralized with Dowex 50 W×2 (H⁺ form, substituted with methanol).The resin was removed by filtration, and the filtrate was concentratedto dryness to give a crude product.

ESIMS: m/z 1222 [M+Na]⁺

Liquid ammonia (about 25 mL) was reservoired at −50° C. in an egg-planttype flask containing the crude product, and 732 mg of metallic sodiumwas added thereto at −50° C. The mixture was vigorously stirred with aglass stirrer bar for 2 hr. Methanol was gradually added until the colorof radicals disappeared. The mixture was returned to room temperature toevaporate ammonia and was finally concentrated to dryness by anevaporator. Water (32 mL) was added to the residue, and the solution wasadjusted to pH 4 to 5 with Dowex 50 W×2 (H⁺ form), and the contents ofthe flask including the resin as such were added to a column packed with10 mL of the same resin. The column was washed with 120 mL of water, andelution was carried out with 1 M aqueous ammonia. A ninhydrin-positivefraction was concentrated to dryness to give a crude product. The crudeproduct was brought to an aqueous solution (40 mL). The aqueous solutionwas charged into a CM-Sephadex C-25 column (equilibrated with 0.005 Maqueous ammonia, 20 mL), and the column was washed with water (40 mL).Elution was carried out with 0.05 M→0.2 M→0.5 M aqueous ammonia (each 40mL), and the corresponding fraction was concentrated to dryness to givethe title compound (104 mg, 43%, as monocarbonate monohydrate).

Rf value: 0.15 (1-butanol:ethanol:chloroform:17% aqueousammonia=4:7:2:7).

ESIMS: m/z 468 [M+H]⁺, 490 [M+Na]⁺

¹H-NMR (26% ND₃-D₂O): δ 5.03, (d, 1H, J=4 Hz), 4.92 (d, 1H, J=3 Hz),4.51 (t, 1H, J=2, 2 Hz), 4.08 (m, 1H), 3.85 (d, 1H, J=2 Hz), 3.80 (m,1H), 3.68 (d, 2H, J=5.5 Hz), 3.60 (dd, 1H, J=4, 10.5 Hz), 3.49 (dd, 1H,J=2, 10 Hz), 3.36 (dd, 1H, J=2, 10 Hz), 3.14 (t, 1H, J=10, 10 Hz), 3.12(t, 1H, J=10, 10 Hz), 3.10 (t, 1H, J=10, 10 Hz), 2.98 (dd, 1H, J=3, 10.5Hz), 2.79 (m, 1H), 2.64 (dd, 1H, J=4, 13.5 Hz), 2.60 (dd, 1H, J=7.5,13.5 Hz), 1.64-1.77 (m, 3H), 1.36 (m, 1H).

¹³C-NMR (26% ND₃-D₂O): δ 102.91, 96.82, 82.89, 76.46, 75.24, 73.81,71.40, 71.07, 70.02, 68.09, 62.70, 54.36, 53.70, 52.49, 50.87, 46.51,28.95, 27.61.

Reference Example 1 Synthesis of(S)-2-benzyloxy-4-benzyloxycarbonylaminobutyric acid

(S)-4-Benzyloxycarbonylamino-2-hydroxybutyric acid (5.25 g, 20.7 mmol)synthesized according to the report of Kawaguchi et al. (Journal ofAntibiotics, Vol. 25, pp. 695-708 (1972)) was dissolved in 60 mL ofN,N-dimethylformamide. Barium oxide (15.9 g, 103.7 mmol) was added underan ice bath. After the elapse of 2 min from the addition of bariumoxide, barium hydroxide octahydrate (13.4 g, 42.5 mmol) was addedthereto under an ice bath. After 10 min, benzyl bromide (7.5 mL, 63.1mmol) was added thereto under an ice bath. After 5 min, the reactionsolution was returned to room temperature and was vigorously stirred.After 1.5 hr, the completion of the reaction was confirmed by TLC(developing solvent system=chloroform:ethyl acetate:acetic acid=10:5:1),and water (0.5 mL) was added thereto under an ice bath. The mixture wasdiluted with chloroform (165 ml), and 4 N HCl (70 mL) was added thereto.Further, water (25 mL) was added followed by separation. The water layerwas extracted with chloroform (50 mL). The organic layer and theextracted organic layer were combined and were washed with a saturatedaqueous NaCl solution. The washed solution was dried over magnesiumsulfate. The solvent was removed by distillation under the reducedpressure, and the residue was purified by column chromatography onsilica gel (silica gel 60, neutral spheres, 160 g) (chloroform:ethylacetate=4:1→chloroform:methanol=10:1→chloroform:methanol:aceticacid=10:1:0.1 (600 mL)) to give(S)-2-benzyloxy-4-benzyloxycarbonylaminobutyrylic acid (2.86 g, 8.34mmol).

LCMS: m/z 344 [M+H]⁺

¹H-NMR (DMSO-d6): δ 12.8 (s, 1H), 7.25-7.35 (m, 10H), 7.26 (d, 1H, J=4.2Hz), 5.01 (s, 2H), 4.49 (ABq, 2H, Jgem=12 Hz), 3.97 (dd, 1H, J=3.7, 8.8Hz), 3.08-3.18 (m, 2H), 1.67-1.93 (m, 2H).

Reference Example 2 (S)-2-Acetoxy-3-amino-N-benzyloxycarbonylpropanoicacid

Step 1 (S)-3-Amino-N-benzyloxycarbonyl-2-hydroxypropanoic acid diphenylmethyl ester

N-Benzyloxycarbonylisoserine (20 mg) synthesized according to the reportof R. D. Westland et al. (Carbohydr. Res., Vol. 28, pp. 268-280 (1973))was dissolved in 0.6 mL of tetrahydrofuran. Diphenylmethylazide (24 mg)was added to the solution, and the mixture was stirred at roomtemperature for 2 hr. The reaction solution was concentrated under thereduced pressure, and the residue was purified by column chromatographyon silica gel (2 g, hexane:ethyl acetate=5:1→4:1→3:1) to give the titlecompound (33 mg, quantitative).

Rf value: 0.54 (hexane:ethyl acetate=1:1)

¹H-NMR (CDCl₃): δ 7.25-7.35 (m, 15H), 6.91 (s, 1H), 5.05 (br, 2H), 4.39(s, 1H), 3.68-3.71 (m, 1H), 3.49-3.58 (dd, 1H, J=6.1, 6.9 Hz), 3.21 (m,1H)

Step 2 (S)-2-Acetoxy-3-amino-N-benzyloxycarbonylpropanoic acid

The compound (26.3 mg) produced in production step 1 was dissolved in1.0 mL of methylene chloride. Pyridine (70 μL) was added to thesolution. Acetic anhydride (20 μL) and 0.8 mg of 4-dimethylaminopyridinewere added thereto at 0° C. The reaction mixture was returned to roomtemperature and was stirred for 2.5 hr. Methanol (100 μL) was addedthereto, and the mixture was stirred at room temperature for 20 min tocomplete the reaction. Chloroform (20 mL) was added thereto, and themixture was washed with 15 mL of water, was dried over a Glauber's saltand was concentrated to dryness to give(S)-3-amino-2-acetoxy-N-benzyloxycarbonylpropanoic acid diphenyl methylester (28.6 mg (99%)).

Rf value: 0.62 (hexane:ethyl acetate=1:1)

¹H-NMR (CDCl₃): δ 7.25-7.37 (m, 15H), 6.87 (s, 1H), 5.26 (dd, 1H, J=4.8,5.2 Hz), 5.06 (br, 2H), 4.93 (s, 1H), 3.66-3.75 (m, 2H), 2.11 (s, 3H).

The above compound (28.6 mg) was dissolved in 0.6 mL of chloroform.Trifluoroacetic acid (0.6 mL) was added to the solution at 0° C., andthe mixture was stirred at room temperature for 2 hr. The reactionmixture was subjected to azeotropic distillation three times withtoluene under the reduced pressure to dryness to give the title compound(21.2 mg) as a crude product.

Rf value: 0.44 (chloroform:ethyl acetate:acetic acid=10:5:1).

¹H-NMR (CDCl₃): δ 7.26-7.35 (m, 5H), 5.25 (dd, 1H, J=4.8, 5.1 Hz), 5.09(br, 2H), 4.86 (s, 1H), 3.62-3.70 (m, 2H), 2.12 (s, 3H).

Reference Example 3 (S)-5-Amino-2-benzyloxy-N-benzyloxycarbonylpentanoicacid

(S)-5-Amino-N-benzyloxycarbonyl-2-hydroxypentanoic acid (21 mg)synthesized according to the report of R. D. Westland et al. (Carbohydr.Res., Vol. 28, pp. 268-280 (1973)) was dissolved in 0.24 mL ofN,N-dimethylformamide. Barium oxide (64 mg) was added to the solutionunder ice cooling, and the mixture was stirred for 2 min. Next, bariumhydroxide (5.4 mg) was added thereto, and the mixture was furtherstirred at the same temperature for 10 min. Benzyl bromide (30 μL) wasadded thereto. After the elapse of 5 min from the start of stirring, thereaction mixture was returned to room temperature, and the mixture wasvigorously stirred for 1.5 hr. Water (15 mL) was added under icecooling, and the mixture was diluted with 30 mL of chloroform. 4 N HCl(0.28 mL) was added to the diluted solution, and the mixture was stirredfor 5 min. The reaction solution was separated, and the water layer wasagain extracted with 20 mL of chloroform. The organic layer and theabove organic layer were combined and were dried over magnesium sulfate.The solution thus obtained was concentrated under the reduced pressure,and the residue was purified by column chromatography on silica gel (30g, hexane:ethyl acetate=4:1→chloroform:methanol=10:1) to give the titlecompound (11 mg, 39%).

Rf value: 0.46 (chloroform:ethyl acetate:acetic acid=10:5:1).

¹H-NMR (CDCl₃): δ 7.32-7.35 (m, 10H), 5.08 (br, 2H), 4.70 (d, 1H, J=11.5Hz), 4.46 (d, 1H, J=11.5 Hz), 4.00 (t, 1H, J=4.99 Hz), 3.19 (br, 2H),1.84 (dt, 2H, J=4.99, 7.0 Hz), 1.66 (tt, 2H, J=7.0, 7.2 Hz).

Reference Example 4 (S)-6-Amino-2-benzyloxy-N-benzyloxycarbonylhexanoicacid

(S)-6-Amino-N-benzyloxycarbonyl-2-hydroxyhexanoic acid (450 mg)synthesized according to the report of R. D. Westland et al. (Carbohydr.Res., Vol. 28, pp. 268-280 (1973)) was dissolved in 5.4 mL ofN,N-dimethylformamide. Barium oxide (1230 mg) was added under icecooling, and the mixture was stirred for 2 min. Next, barium hydroxide(1061 mg) was added thereto, and the mixture was further stirred at thesame temperature for 10 min. Thereafter, 0.57 mL of benzyl bromide wasadded. After the elapse of 5 min from the addition of benzyl bromide,the mixture was returned to room temperature, and the mixture wasvigorously stirred for 1.5 hr. Water (15 mL) was added thereto under icecooling. The mixture was diluted with 30 mL of chloroform. 4 N HCl (6.0mL) was added thereto, and the mixture was stirred for 5 min. Thereaction solution was separated by filtration. The water layer was againextracted with 25 mL of chloroform. The organic layer and the aboveorganic layer were combined and were dried over magnesium sulfate. Thesolution was concentrated under the reduced pressure, and the residuewas purified by column chromatography on silica gel (32 mg, hexane:ethylacetate=3:1) to give the title compound (196 mg, 33%).

Rf value: 0.47 (chloroform:ethyl acetate:acetic acid=10:5:1)

¹H-NMR (CDCl₃): d 7.29-7.34 (m, 10H), 5.09 (br, 2H), 4.74 (d, 1H, J=11.7Hz), 4.46 (d, 1H, J=11.7 Hz), 3.97 (t, 1H, J=5.7 Hz), 3.16 (br, 2H),1.77-1.83 (m, 2H), 1.41-1.50 (m, 4H).

Reference Example 5 (R)-2-Benzyloxy-4-benzyloxycarbonylaminobutyric acid

4-Benzyloxycarbonylamino-2-(R)-hydroxybutyric acid (2.245 g) synthesizedaccording to the method of H. Naito et al. (J. Antibiot., Vol. 26, pp.297-301 (1973)) was dissolved in 25.6 mL of N,N-dimethylformamide.Barium oxide (6.795 g) was added to the solution under ice cooling withstirring, and the mixture was vigorously stirred for 2 min. Bariumhydroxide octahydrate (5.593 g) was added to the reaction mixture, andthe mixture was vigorously stirred under ice cooling for 10 min. Benzylbromide (3.16 mL) was added thereto, and the mixture was vigorouslystirred under ice cooling for 5 min. The reaction mixture was returnedto room temperature and was vigorously stirred for 1.5 hr. Water (0.48mL) was added under ice cooling, and the mixture was diluted with 70 mLof chloroform. 4 M hydrochloric acid (31 mL) was added to the dilutedsolution. Water (11 mL) was then added thereto followed by separation.The water layer was extracted with 22 mL+10 mL of chloroform. Thecombined organic layers were washed with 30 mL of saturated brine, weredried over magnesium sulfate and were concentrated to dryness to give4.54 g of a crude product. The product was purified by columnchromatography on 70 g of a neutral silica gel(chloroform→chloroform:ethylacetate=4:1→chloroform:methanol=10:1→chloroform:methanol-aceticacid=10:1:0.1) to give the title compound (1.107 g, 36%).

Rf value: 0.57 (chloroform:ethyl acetate:acetic acid=10:5:1)

ESIMS: m/z 342 [M−H]⁻

¹H-NMR (CDCl₃): δ 7.25-7.40 (10H), 5.06 (s, 2H), 4.94 (br. s, 1H),4.40-4.76 (ABq, 2H, Jgem=11 Hz), 4.06 (t, 1H, J=5.5, 5.5 Hz), 3.35 (m,2H), 2.02 (m, 2H).

Test Example 1 Antimicrobial Activity

MIC of the compound represented by formula (I) produced in Example 1against an MRSA strain, specifically a clinically isolated MRSA strain(n=9), which has low sensitivity to arbekacin and against which theminimal inhibitory concentration (MIC, μg/mL) is 4 to 8, was measured byan agar plate dilution method according to the standard method of JapanSociety of Chemotherapy (Chemotherapy, Vol. 29, pp. 76 to 79, 1981).

As a result, the compound represented by formula (I) has an MIC value of0.5 to 2. The compound represented by formula (I) had higherantimicrobial activity than arbekacin, against the above MRSA strainhaving low sensitivity to arbekacin.

Test Example 2 Antimicrobial Activity

MIC of the 2-hydroxyarbekacins produced in Examples 1 to 4 and thecompounds produced in Examples 5, 6, 8, 9, 10, and 11, against an MRSAstrain, specifically a clinically isolated MRSA strain, which isdifferent from that used in Test Example 1 and has high resistance toarbekacin and against which the MIC value (μg/mL) is 128, was measuredby an agar plate dilution method according to the standard method ofJapan Society of Chemotherapy (Chemotherapy, Vol. 29, pp. 76 to 79,1981).

As a result, these compounds had an MIC value of 2 to 32, indicatingthat the compounds represented by formula (I) had higher antimicrobialactivity than arbekacin, against the MRSA strain showing high resistanceto arbekacin.

Test Example 3 Evaluation of Influence on Kidney of Normal Mice

An evaluation system of influence on the kidney was constructed based onrenal disease model preparation method using gentamicin (KIDNEY ANDDIALYSIS, Vol. 31, 1991, extra edition, “Renal Disease Model”, p. 423“Drug-induced Renal Damage and its Examination Method,” Kidney andDialysis Editorial Committee, published by TOKYOIGAKUSYA). The influenceof the compound represented by formula (I) produced in Example 1 on thekidney was evaluated using this evaluation system. A physiologicalsaline administration group and an arbekacin administration group wereset as control groups. Each group consisted of four mice (Crj: CD-1(ICR) female mice of eight weeks old).

In the above evaluation system, the evaluation of nephrotoxicity wascarried out using NAG as an index. NAG exists in lysosome in each tissueof humans and animals, is an enzyme, which converts a mucopolysaccharideto a glycoprotein, for example, converts β-N-acetyl-D-glucosaminide toβ-N-acetyl-D-glucosamine, and is plentifully present in proximaluriniferous tubule epithelial cells of the kidney in whichaminoglycoside antibiotics are particularly accumulated. When thekidney, especially its proximal uriniferous tubule is damaged, NAG isreleased into urine to increase the amount of NAG in the urine.Accordingly, the increase in the amount of NAG in urine is considered toreflect proximal uriniferous tubule damage and is adopted as one ofbiochemical markers for estimating the level of renal insufficiency inclinical test items. The amount of NAG is measured by an examinationmethod, for example, an MCP-NAG method (Outline of Latest InternalMedicine, Vol. 4, Clinical Laboratory—Kensa No Susumekata To Deta NoYomikata (How To Forward Examination and How To Read Data)—<Naika Soron4 (General Remarks Of Internal Medicine 4> p. 274, Imura et al. (ed.),published by Nakayama Syoten Co., Ltd., 1994).

A solution of physiological saline of the compound according to thepresent invention having a concentration of 12 mg/mL was administered tothe mice in each group at a dose of 120 mg/kg/day (in two divided doses;morning and afternoon) for 4 days by repeated intraperitonealadministration. Naturally excreted urine was collected for about 24 hrfrom the completion of the administration in the morning on day 4, andthe amount of NAG in the urine was measured by the MCP-NAG method. Thesame treatment was carried out for the physiological salineadministration group and the arbekacin administration group.

The results were as shown in Table 1. Table 1 shows the average value ofthe amount of NAG for each group.

TABLE 1 Influence on kidney of normal mice Compound administered Amountof NAG in urine (mIU) Compound represented 74.7 by formula (I) Arbekacin135.0 Physiological saline 19.7

As described in Table 1, the amount of NAG in the urine for the group towhich the compound represented by formula (I) had been administered, wassmaller than the amount of NAG for the group to which arbekacin has beenadministered.

Reference Test Example 1

The influence of arbekacin and an arbekacin analogue TS2037(5,4″-diepiarbekacin) produced according to the description of WO2005/070945 on the kidney was evaluated in the same manner as in TestExample 3. As a result, the amount of NAG in the urine for the TS2037administration group was 358.8 (mIU), whereas the amount of NAG in theurine for the arbekacin administration group was 168.5 (mIU). That is,the NAG amount for arbekacin analogue TS2037 was higher than that forarbekacin.

1-36. (canceled)
 37. A compound represented by formula (Xa):

wherein R^(5ax) and R^(5eq), which are different from each other,represent a hydrogen atom or hydroxyl.
 38. A compound represented byformula (XXV):

wherein R² and G represent a protective group for hydroxyl, R³, R^(2′),R^(6′) and E represent a protective group for amino, n is an integer of1 to 4, and the steric configuration of carbon atom attached with *represents R or S, or its diastereomer mixture with respect to thecarbon atom attached with *.
 39. The compound according to claim 38 orits diastereomer mixture with respect to the carbon atom attached with*, wherein R² represents optionally substituted aryl C1-3 alkyl, R³,R^(2′) and R^(6′), which may be the same or different, representoptionally substituted C1-6 alkylsulfonyl, optionally substitutedarylsulfonyl, or optionally substituted C1-6 alkyloxycarbonyl, Erepresents optionally substituted C1-6 alkyloxycarbonyl, and Grepresents a hydrogen atom, optionally substituted C1-6 alkylcarbonyl,optionally substituted arylcarbonyl, or optionally substituted aryl C1-3alkyl.
 40. The compound according to claim 38 or its diastereomermixture with respect to the carbon atom attached with *, wherein R²represents aryl C1-3 alkyl optionally substituted by methoxy or nitro,R³, R^(2′) and R^(6′), which may be the same or different, representC1-6 alkylsulfonyl optionally substituted by optionally substitutedphenyl, arylsulfonyl optionally substituted by methyl, or C1-6alkyloxycarbonyl optionally substituted by optionally substitutedphenyl, E represents C1-6 alkyloxycarbonyl optionally substituted byoptionally substituted phenyl, and G represents a hydrogen atom, C1-6alkylcarbonyl, arylcarbonyl, or aryl C1-3 alkyl optionally substitutedby methoxy.
 41. The compound according to claim 38 or its diastereomermixture with respect to the carbon atom attached with *, wherein R²represents benzyl, methoxybenzyl or nitrobenzyl, R³, R^(2′) and R^(6′),which may be the same or different, represent methanesulfonyl,benzylsulfonyl, p-toluenesulfonyl, benzyloxycarbonyl,tert-butoxycarbonyl, p-methoxybenzyloxycarbonyl, orp-nitrobenzyloxycarbonyl, E represents benzyloxycarbonyl, and Grepresents a hydrogen atom, acetyl, benzoyl, benzyl, p-methoxybenzyl, ortriphenylmethyl.
 42. A compound represented by formula (Xb):

wherein R^(5 ax) and R^(5eq), which are different from each other,represent a hydrogen atom or hydroxyl, and R^(4″ax) and R^(4″eq), whichare different from each other, represent a hydrogen atom or hydroxyl.43. A compound represented by formula (XIV):


44. A process for producing a compound represented by formula (Ia):

wherein R^(5ax) and R^(5eq), which are different from each other,represent a hydrogen atom or hydroxyl, and R^(4″ax) and R^(4″eq), whichare different from each other, represent a hydrogen atom or hydroxyl, nis an integer of 1 to 4, and the steric configuration of carbon atomattached with * represents R or S, the process comprising the steps ofintroducing protective groups into amino groups in a compoundrepresented by formula (Xa):

wherein R^(5ax) and R^(5eq) are as defined in formula (Ia), reacting thecompound represented by formula (Xa) with a compound represented byformula (Xc):

wherein W represents a leaving group; Y^(ax) and Y^(eq), which may bethe same or different, represent group —OR^(4″) or a hydrogen atom;R^(2″), R^(4″) and R^(6″) represent a protective group for hydroxyl; andthe steric configuration of carbon atom attached with * represents R orS, removing the protective group from the resultant compound andconverting an azido group in the compound to an amino group to give acompound represented by formula (Xb):

wherein R^(5ax), R^(5eq), R^(4″ax) and R^(4″eq) are as defined informula (Ia), optionally introducing protective groups into functionalgroups other than the amino group at 1-position of the compoundrepresented by formula (Xb), reacting the resultant compound with acompound represented by formula (XVII):

wherein E represents a protective group for amino group; G represents aprotective group for hydroxyl group; F represents a hydrogen atom or acarboxylic acid activating group; n is an integer of 1 to 4; and thesteric configuration of carbon atom attached with * represents R or S,and removing the protective groups of the resultant compound to give thecompound represented by formula (Ia).
 45. The process according to claim44, wherein R^(5ax) represents a hydrogen atom, and R^(5eq) representshydroxyl.
 46. The process according to claim 44, wherein R^(5ax)represents hydroxyl, and R^(5eq) represents a hydrogen atom.
 47. Aprocess for producing a compound represented by formula (Ia):

wherein R^(5ax) represents a hydrogen atom, R^(5eq) represents hydroxyl,R^(4″ax) and R^(4″eq), which are different from each other, represent ahydrogen atom or hydroxyl, n is an integer of 1 to 4, and the stericconfiguration of carbon atom attached with * represents R or S, theprocess comprising the steps of introducing protective groups into aminogroups at the 3-, 2′- and 6′-positions and the hydroxyl group at2-position of a compound represented by formula (Xa):

wherein R^(5ax) and R^(5eq) are as defined in formula (Ia), reacting theresultant compound with a compound represented by formula (XVII):

wherein E represents a protective group for amino group; G represent aprotective group for hydroxyl group; F represents a hydrogen atom or acarboxylic acid activating group; n is an integer of 1 to 4; and thesteric configuration of carbon atom attached with * represents R or S,to give a compound represented by formula (XXV):

wherein R² represents a protective group for hydroxyl group; R³, R^(2′)and R^(6′) represent a protective group for amino group; and E, G, n andthe steric configuration of the carbon atom attached with * are asdefined in formula (XVII), reacting the compound represented by formula(XXV) with a compound represented by formula (Xc) or (Xd):

wherein W represents a leaving group; Y^(ax) and Y^(eq), which aredifferent from each other, represent group —OR^(4″) or a hydrogen atom;R^(2″), R^(4″) and R^(6″) represent a protective group for hydroxylgroup; and the steric configuration of carbon atom attached with *represents R or S,

wherein W, Y^(ax), Y^(eq), R^(2″), R^(6″), and the steric configurationof carbon atom attached with * are as defined in (Xc); and R^(3″)represents a protective group for amino group, and removing theprotective groups from the resultant compound and, when the compoundrepresented by formula (Xc) is used, converting an azido group in thecompound to an amino group, to give a compound represented by formula(Ia).
 48. The process according to claim 45, wherein the compoundrepresented by formula (Xa) is produced by hydrolyzing the compoundrepresented by formula (II):


49. The process according to claim 46, wherein the compound representedby formula (Xa) is produced by introducing protective groups intohydroxyl groups other than those located at 4″- and 5-positions andamino groups in a compound represented by formula (II):

reversing the steric configuration of the hydroxyl group at the5-position of the resultant compound, and removing the protective groupfrom the resultant compound, and further hydrolyzing the compound togive the compound represented by formula (Xa).
 50. The process accordingto claim 49, which further comprises, before or simultaneously with thereversing of the steric configuration of the hydroxyl group at5-position, eliminating the hydroxyl group at 4″-position.
 51. Theprocess according to claim 47, wherein the compound represented byformula (Xa) is produced by hydrolyzing the compound represented byformula (II):